The cerulenin-induced formation of 1-acyl-lysophosphatidyl glycerol in Bacillus megaterium.

When a culture of $\text{Bacillus megaterium}$ ATCC 14581, growing at 20° and treated with the fatty acid synthesis inhibitor, cerulenin, was incubated with [U-¹⁴C]palmitate, 50% of the incorporated label was found in 1-palmitoyl-lysophosphatidyl glycerol within 5 min. Most of the remaining ¹⁴C appeared in free fatty acid and phosphatidyl glycerol. By 45 min almost all of the lyso compound had disappeared and 80% of the incorporated label was found in phosphatidyl glycerol. At 20°, in the absence of cerulenin or at 35° in either its presence or absence, no labeled lysophosphatidyl glycerol could be found at any time after [U-¹⁴C]palmitate addition. The major radioactive lipid, in these cases, was always phosphatidyl glycerol. At 20°, the palmitate of phosphatidyl glycerol but not of lysophosphatidyl glycerol was readily desaturated.     

Displacement of palmitate from albumin by chlorophenoxyisobutyrate.

The effect of chlorophenoxyisobutyrate, a hypolipidemic drug that decreases plasma free fatty acids, triglycerides, and cholesterol, on the partitioning of [¹⁴C]-palmitate between hexane and bovine serum albumin was studied at 37°. In this system, hexane served as a hydrophobic trap for free fatty acids displaced from BSA by chlorophenoxyisobutyrate, allowing less than 0.3% to remain in the aqueous phase. As the concentration of chlorophenoxy isobutyrate was raised from 0.4 to 3.2 mM, there was a progressive displacement of palmitate from the [¹⁴C]-palmitate-BSA complex into hexane, the magnitude being dependent on the initial $\text{V}$ value (moles palmitate bound/mole BSA). Beginning with [¹⁴C]-palmitate in hexane, chlorophenoxyisobutyrate (2 mM) decreased the moles palmitate bound/mole of BSA by 16% at $\text{V}$ = 0.2, and 34% at $\text{V}$ = 3.0.     

DIFFERENTIAL DISTRIBUTION OF [U-14C]GLUCOSE AND [U-14C]GLYCEROL AMONG MOLECULAR SPECIES OF PHOSPHATIDYL CHOLINE,PHOSPHATIDYL ETHANOLAMINE AND 1,2-DIACYLGLYCEROL IN RABBIT BRAIN12

Specific radioactivities of molecular species of phosphatidyl choline(PC), phosphatidyl ethanolamine(PE) and 1,2-diacylglycerol were determined in rabbit brain 15 and 30 min after intraventricular injection of 10OpCi of either [U-¹⁴C]glucose or [U-¹⁴C]glycerol. The rate of de nouo synthesis of glycerophospholipids and their molecular species could be determined after glycerol labelling, since 94.0–99.7% of ¹⁴C activity was recovered in glyceryl moieties of brain lipids. After injection of glucose radioactivity was measured in both glyccrol and acyl residues of lipids. High incorporation rates were measured in species of PC, PE and 1,2-diacylglycerol with oleic acid in position 2 and with palmitic, stearic or oleic acids in position 1. The conclusion may therefore be drawn that these molecular species were preferably synthesized de novo by selective acylation of glycerol 3-phosphate. The lowest specific activities were observed for 1,2-dipalmitoyl- and l-stearoyl-2- arachidonoyl-glycerol, -PC and -PE. These turnover rates point to incorporation of arachidonate, and probably also of palmitate in dipalmitoyl-PC, amounting to 20% of total PC, via deacylation-acylation- cycle.     

An apolipoprotein preferentially enriched in cholesteryl ester-rich very low density lipoproteins

Phosphatidyl glycerol is present in lamellar bodies and in the material obtained by alveolar wash representing 12.3 and 11.5%, respectively, of the total phospholipid phosphorus. Lung microsomes catalyze the formation of phosphatidyl glycerol from the known precursors, L-glycerol 3-phosphate and CDP-diglyceride. The rate of [¹⁴C]L-glycerol 3-phosphate incorporation into phosphatidyl glycerol was 30% higher in microsomes as compared to mitochondria. The addition of mercuric chloride inhibited the synthesis of phosphatidyl glycerol, and stimulated the incorporation into another as yet incompletely identified lipid. After pulse labeling of microsomal phosphatidyl glycerol $\text{in vitro}$, further incubation of microsomes with lamellar bodies or alveolar wash resulted in nearly quantitative appearance of label in surfactant.     

In vivo biosynthesis of -linolenic acid in plants

[1-¹⁴C]acetate was readily incorporated into unsaturated fatty acids by leaf slices of spinach, barley and whole cells of $\text{Chlorella}\text{pyrenoidosa}$ and $\text{Candida}\text{bogoriensis}$. In these systems the [¹⁴C] label in newly synthesized oleate and linoleate was approximately equally distributed in the C_1–9 and the C_10–18 fragments obtained by reductive ozonolysis of these acids, whereas in a-linolenic acid over 90% of the total [¹⁴C] was localized in the C_1–9 fragment. While [1-¹⁴C]oleic acid was converted by whole cells of $\text{Chlorella}$ to [1-¹⁴C]linoleic and [1-¹⁴C]linolenic acids, [U-¹⁴C]oleic acid yielded [U-¹⁴C]linoleic acid but a-linolenic acid was labeled only in the carboxyl terminal carbon atoms. When spinach leaf slices were supplied with carboxyl labeled octanoic, decanoic, dodecanoic, tetradecanoic and octadecanoic acids, only the first three acids were converted to a-linolenic acids while the last two acids were ineffective. Thus we suggest that (a) linoleic acid is not the precursor of a-linolenic acid and (b) 12:3(3, 6, 9) is the earliest permissible trienoic acid which is then elongated to a-linolenic acid.     

The occurrence of phosphatidyl choline exchange protein in leaves

The transfer of phosphatidyl choline between liposomes was stimulated by the protein fractions from spinach leaves, etiolated and greening leaves of $\text{Avena}$ seedlings. This is confirmed by the transfer of [¹⁴C]phosphatidyl choline or spin-labeled phosphatidyl choline between donor and acceptor liposomes. ESR spectrum changes also indicated that no spin-labeled phosphatidyl choline was released from donor liposomes by spinach leaf protein unless acceptor liposomes were present. [¹⁴C]phospholipids were transferred from liposomes to both spinach chloroplasts and $\text{Avena}$ etiochloroplasts by phosphatidyl choline exchange protein from germinated castor bean endosperms and further from liposomes to spinach chloroplasts by spinach leaf protein. These results support the view that phosphatidyl choline in the plastid is supplied from the synthesis site, the endoplasmic reticulum, by phospholipid exchange protein.     

Characterization of particulate D-apiosyl-and D-xylosyltransferase from Lemna minor.

A particulate enzyme preparation capable of catalyzing the transfer of d-[U-¹⁴C]apiose and d-[U-¹⁴C]xylose from uridine 5′-(α-d-[U-¹⁴C]apio-d-furanosyl pyrophosphate) (UDP[U-¹⁴C]Api) and uridine 5′-(α-d-[U-¹⁴C]xylopyranosyl pyrophosphate) (UDP[U-¹⁴C]Xyl) to endogenous acceptor molecules was isolated from Lemna minor. The two enzymes were named UDP-d-apiose:acceptor d-apiosyltransferase and UDP-d-xylose:acceptor d-xylosyltransferase and were associated with particulate material sedimenting between 480 and 34,800g. The rate of d-[U-¹⁴C]apiose or d-[U-¹⁴C]xylose incorporation was proportional to the quantity of enzyme preparation used and was constant with time to 1.5 min. Both enzymes showed a pH optimum of 5.7 in citrate-phosphate buffer. The d-apiosyltransferase has a K_m for UDP[U-¹⁴C]Api of 4.9 μm. Bovine serum albumin and sucrose stimulated the rate of incorporation of both pentoses. Both enzymes rapidly lost activity; with our best conditions, approximately 50% of each enzyme activity was lost in 6 min at 25 °C or in 3 h at 4 °C. Incorporation of d-[U-¹⁴C]apiose was obtained in the absence of added uridine 5′-(α-d-galactopyranosyluronic acid pyrophosphate) (UDPGalUA); however, the addition of UDPGalUA not only almost doubled the rate of incorporation, but also increased the total incorporation of d-[U-^l4C]apiose and extended the proportional range of incorporation at 25 °C from 1.5 to 2 min.     

The time course of glucagon action on the utilization of [U-14C]palmitate by isolated hepatocytes

The time course of glucagon action on the utilization of [U-¹⁴C]palmitate by isolated hepatocytes was studied. Ten minutes incubation of the cells after hormone addition was required in order to observe increased oxidation and decreased esterification of the labeled palmitate. The acid-soluble, labeled oxidation products could be separated into two main fractions, glucose and ketone bodies. Initially, glucagon directed the flux of radioactivity toward glucose and CO₂. After prolonged incubation in the presence of glucagon, labeled ketone bodies, as well as labeled glucose and ¹⁴CO₂, were increased. This effect was most marked as regards glucose. The results indicate that glucagon induces a rapidly onset stimulation of the rates of Krebs cycle and gluconeogenesis, while increased oxidation and decreased esterification of palmitate are time-delayed corresponding to the establishment of a lower level of glycerophosphate. About 10% of the glucose carbon formed by gluconeogenesis originated from the fatty acid when cells from fasted rats were incubated in the presence of alanine and [U-¹⁴C]palmitate.     

Effect of temperature of imbibition on phospholipid metabolism in pea embryonic axes

Pea seeds were imbibed in radioactive choline and then germinated. Treatments were either at 5° or 25° and the seeds were imbibed for 5 hr at one temperature and then transferred to the other. [$\text{Me}$-¹⁴C]Choline incorporation into phosphatidyl choline in the ER and the plasma membrane obtained from the embryonic axes after germination was measured. Seeds kept constantly at 25° had a very rapid initial incorporation of choline followed by a loss of label. Seeds kept at 5° had a very much lower rate of incorporation. However, seeds transferred from 5 to 25° behaved for at least 48 hr as if continuously kept at 5°, while in seeds transferred from 25 to 5° incorporation stopped after 15 hr. The seeds apparently respond to transient exposure to temperature by a changed metabolism of phospholipid. Data are also given for the choline content of the seeds under the different treatments and for the changes in total phospholipid.     

Biosynthesis and metabolism of steryl glycosides in Sinapis alba seedlings

With ¹⁴CO₂, d-glucose-[U-¹⁴C] and dl-mevalonate-[4R-4-³H₁] used as precursors, a study was made of the labelling dynamics of the steryl glucosides (SG) and steryl acylglucosides (ASG) in Sinapis alba seedlings. The radioactivity of the sterol and sugar moieties, as well as of the fatty acid moieties in the case of ASG, was analysed separately. The course of incorporation of ¹⁴C from ¹⁴ CO₂ and glucose-[U-¹⁴C] into the sugar part of SG and ASG indicated that about $\text{2}\text{3}$ of the whole pool of the newly synthesized sterol glycosides of both types underwent rapid deglucosylation. Likewise, fatty acids in the ASG pool were rapidly exchanged. The present results point to a high metabolic activity of the sterol glycoside derivatives in plant cells.     

A new in vivo metabolite of vitamin D3: 1,25,26-trihydroxyvitamin D3

The enzymatic conversion of 5-methylthioribose to methionine and its deaminated derivatives, 2-keto-4-methylmercaptobutyric acid and 2-hydroxy-4-methylmercaptobutyric acid by cell-free extracts of $\text{Enterobacter}\text{aerogenes}$ has been demonstrated. ¹⁴C-Labeled methionine was isolated from incubation mixtures with 5-methylthio[U-¹⁴C]ribose. The carbohydrate part of this compound furnishes at least part, if not all, of the four carbon chain of methionine.     

The influence of temperature and gamma-aminobutyric acid on benzodiazepine receptor subtypes in the hippocampus of the rat

The influence of GABA on the affinity of flunitrazepam (FLU) for benzodiazepine receptor subtypes (type I and II) was studied by measurement of the competitive inhibition of [³H]FLU and [³H]propyl beta-carboline-3-carboxylate ([³H]PCC) binding. When assays were carried out at 0°C using a low concentration (0.040 nM) of [³H]PCC so that the type I receptors were selectively labelled, no significant effect of GABA (10^?4 M) on the $\text{FLU}\text{[}{}^3\text{H]}\text{PCC}$ competition curve was detected. In contrast, when assays were carried out at 0°C using [³H]FLU or a high concentration of [³H]PCC to achieve [³H]ligand receptor occupancy of both type I and type II receptors, GABA (10^?4 M) caused a significant increase in the affinity of FLU as measured by $\text{FLU}\text{[}{}^3\text{H]}\text{FLU}$ and $\text{FLU}\text{[}{}^3\text{H]}\text{PCC}$ competition experiments. Collectively, these data suggest that the influence of GABA on benzodiazepine receptor binding is mediated, primarily, by the type II receptor. It was also noted that the $\text{PCC}\text{[}{}^3\text{H]}\text{FLU}$ competition curve had a Hill coefficient of approximately 1 at 37°C as compared to the results of experiments at 0°C during which a Hill coefficient of approximately 0.7 was calculated.     

Accumulation of 2-deoxyglucose against its concentration gradient in rat adipocytes

Rat adipocytes were incubated at 37°C with 2-deoxy-d-[1-¹⁴C]glucose ([¹⁴C]2dGlc) at various concentrations and the intracellular concentrations of [¹⁴C]2dGlc and deoxy[¹⁴C]glucose phosphate ($\text{[}{}^{14}\text{C}\text{]2}\text{dGlc}\text{P}$) were measured. Using 7 μM extracellular [¹⁴C]2dGlc, the intracellular [¹⁴C]2dGlc concentration approached the extracellular by 5 min in insulin-stimulated cells and by 60 min it exceeded the extracellular concentration by 50-fold. A maximum accumulation ratio of 3.5 was reached by 7 min using 1 mM and a ratio of 1.6 was reached by 1 to 3 min using 10 mM extracellular 2dGlc. The time at which the concentration of intracellular 2dGlc exceeded the extracellular was inversely related to the accumulation of $\text{2}\text{dGlc}\text{P}$. The rate of accumulation of total radioactivity ([¹⁴C]2dGlc plus $\text{[}{}^{14}\text{C}\text{]2}\text{dGlc}\text{P}$ decreased after 20 min using 7 μM extracellular [¹⁴C]2dGlc. This change occurred later at 22°C or in the absence of insulin and sooner at higher concentrations of 2dGlc. Experiments where uptake was stopped by dilution indicated that radioactivity appearing in the medium was [¹⁴C]2dGlc, but radioactivity disappearing from the cells was largerly $\text{[}{}^{14}\text{C}\text{]2}\text{dGlc}\text{P}$. Addition of 10 mM unlabelled 2dGlc or glucose to cells preincubated with 7 μM [¹⁴C]2dGlc resulted in a more rapid loss of accumulated label from the cells, while addition of 10 mM $\text{3-O-}\text{methylglucose}$, a non-metabolizeable sugar analogue with about the same affinity for the transport system as 2dGlc, was without effect. The results show that 2dGlc is accumulated against its concentration gradient. It is suggested that the mechanism involves first, dephosphorylation of $\text{2}\text{dGlc}\text{P}$ and second, the presence of a diffusion barrier between the site of dephosphorylation and the transport site.     

Phospholipid activation of Lactobacillus plantarum undecaprenyl pyrophosphate synthetase

The ability of a number of phospholipids to stimulate $\text{Lactobacillus}$$\text{plantarum}$ undecaprenyl pyrophosphate synthetase was investigated. The detergent Triton X-100, which is added to stabilize the enzyme during purification and is required for $\text{in}$$\text{vitro}$ activity, was removed with the non-ionic resin XAD-2. The effects of cardiolipin, phosphatidyl ethanolamine, phosphatidyl choline, and phosphatidyl glycerol on the activity of XAD-2 treated undecaprenyl pyrophosphate synthetase were determined. Of the phospholipids studied only cardiolipin stimulated $\text{in}$$\text{vitro}$ enzymic activity as effectively as Triton X-100.     

Characteristics of leucine transport by isolated hepatocytes of antarctic fish at low temperatures

Hepatocytes prepared by collagenase perfusion from Antarctic nototheniid fish of genus Trematomus are active in uptake of [¹⁴C]leucine at 0, 5, and 10°C. The system is saturable with apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ about 1.0 mM. Isoleucine and phenylalanine were major competitors, valine was about one-half as effective, while alanine, glycine and histidine had no effect. Temperature dependency of rates in the 0–10°C range yielded $\text{E}{}_{\mathrm{<mtext>a</mtext>}}\text{= 65}\text{kJ/mol}$ ($\text{Q}{}_{10}\text{= 2.7}$). The average first-order rate constant at 0°C was 0.1 min^?1, one-third the value of 0.3 min^?1 estimated for clearance of [¹⁴C]leucine by liver of these species in vivo. Affinity and specificity agreed well with in vivo data on liver clearance of leucine, both in Antarctic fish at 0°C and in temperate fish acclimated to 10°C and 20°C. The results indicate similar modifications of leucine transport associated with evolutionary cold adaptation and seasonal acclimation in fish.     

Biosynthesis of N-methyl-l-glucosamine from d-glucose by Streptomyces griseus

Biosynthesis of $\text{N-}\text{methyl}\text{-}\text{l}\text{-}\text{glucosamine}$ moiety of streptomycin from d-glucose by Streptomyces griseus was studied. A mixture of d-[1-¹⁴C]glucose and d-[6-³H]glucose was given to the culture of S. griseus. The ³H/¹⁴C ratio found in $\text{N-}\text{methyl}\text{-}\text{d}\text{-}\text{glucosamine}$ further supports a mechanism that the conversion of d-glucose to l-hexose is carried out without scission of carbon skeleton. When d-[1-¹⁴C]glucose and d-[3-³H]glucose were used, the fall of ³H/¹⁴C ratio in $\text{N-}\text{methyl}\text{-}\text{l}\text{-}\text{glucosamine}$ showed that the hydrogen atom at C-3 plays a rôle in such a transformation.     

Ozonolytic cleavage of authentic and pancreatic dolichyl mannopyranosyl phosphates: determination of sugar configuration in the fragments with alpha- and beta-mannosidases.

Exposure of authentic dolichyl α-D-[¹⁴C]mannopyranosyl phosphate ($\text{I}$) or calf pancreas dolichyl [¹⁴C]mannopyranosyl phosphate ($\text{II}$) to ozone at ?70° in pentane followed by treatment with triphenylphosphine gave water-soluble fragments in 65–95% yield. The radioactive products obtained were similar; the major fragment had a mobility on tlc greater than that of mannose but lower than that of citronellyl β-D-mannopyranosyl phosphate. The electrophoretic behavior of the fragments indicated that they possessed intact phosphodiester linkages. α-Mannosidase released [¹⁴C]mannose from the fragments of $\text{I}$ but not from the fragments of $\text{II}$; however, the latter were susceptible to β-mannosidase indicating that the pancreatic mannolipid contains a β-linked mannosyl residue.     

Alternate pathways of linolenic acid biosynthesis in growing cultures of Penicillium chrysogenum

Evidence was obtained that Penicillium chrysogenum can produce linolenate by two biosynthetic pathways, i.e., by elongation of a shorter trienoic acid as well as direct desaturation of 18-C acids. In oxygen deficient cultures, exogenous hexadecatrienoate stimulated [1-¹⁴C]acetate incorporation into labeled octadecatrienoate and [U-¹⁴C]hexadecatrienoate with nonlabeled acetate yielded linolenate that had relatively little label in the 1-C position. With [1-¹⁴C]acetate as the only added substrate, oxygen deficiency inhibited incorporation of label into monoenoic and dienoic acids but not into trienoic acids. Incorporation of the [U-¹⁴C]linoleate into linolenate also was inhibited.In aerated cultures, 1-¹⁴C-label from laurate, palmitate, stearate, oleate, linoleate, and hexadecatrienoate was readily incorporated into linolenate. Decarboxylation and oxidation studies indicated that the longer acids were incorporated largely intact. [U-¹⁴C]Linoleate was incorporated into linolenate in which the fraction of label in 1-C was similar to that of the substrate. These data suggest that this mold has broader synthetic capabilities than do some chloroplast systems for the biosynthesis of linolenate.     

Juvenile hormone production by corpora allta of Tenebrio molitor in vitro

$\text{In vitro}$ organ cultures of corpora allata or corpora cardiaca-corpora allata complexes from $\text{Tenebrio molitor}$ were found to produce methyl-(2E, 6E)-(10R)-10, 11- epoxy-3, 7, 11 trimethyl-2, 6-dodecadienoate (JH III). No detectable JH I or II was produced. The hormone was identified by derivation, chromatography and mass spectral analysis. A ¹⁴C radiolabel was incorporated into the methyl carbon of the ester function from precursor L-[$\text{methyl}$-¹⁴C]-methionine added to the culture medium.     

Enzymatic transfer of the acyl group from acyl dihydroyxacetone phosphate to different substrates

The acyl group of acyl dihydroxyacetone phosphate was shown to be enzymatically transferred in guinea pig liver mitochondria to various acceptors such as lysolecithin, lysophosphatidyl ethanolamine and $\text{sn}$-glycerol-3-phosphate to form lecithin, phosphatidyl ethanolamine and phosphatidate, respectively. Coenzyme A and Mg⁺⁺, but not ATP, were required for this reaction. A rapid exchange of acyl group between acyl dihydroxyacetone phosphate and dihydroxyacetone phosphate was also observed.