The role of the endoplasmic reticulum in the synthesis and transport of α-amylase in barley aleurone layers

The subcellular site of -amylase (EC 1.6.2.1) synthesis and transport was studied in barley aleurone layers incubated in the presence or absence of gibberellic acid (GA₃). Using [³⁵S]methionine as a marker, the site of amino-acid incorporation into organelles isolated from aleurone layers incubated with and without GA₃ was determined following purification by isopycnic sucrose-density-gradient centrifugation. Incorporation of radioactivity into trichloroacetic-acid-insoluble proteins was greatest in those fractions exhibiting activity of an endoplasmic reticulum (ER) marker enzyme. Further fractionation of densitygradient fractions by sodium-dodecyl-sulfate polyacrylamide-gel electrophoresis showed that a major portion of the radioactivity in the ER fractions was present in a protein co-migrating with marker -amylase. This protein was identified as authentic -amylase by immunoadsorbent chromatography and affinity chromatography. The newly synthesized -amylase associated with the ER was shown to be sequenstered within the lumen of the ER by experiments which showed that the enzyme was resistant to proteolytic degradation. The labelled -amylase sequestered in the ER can be chased from this organelle when tissue is incubated in unlabelled methionine following a 1-h pulse of labelled methionine. The isoenzymic forms of -amylase found in tissue homogenates and incubation media of aleurone layers incubated with and without GA₃ were characterized after chromatography on diethylaminoethyl cellulose. In homogenates of GA₃-treated aleurone layers, five peaks of -amylase activity were detected, while in homogenates of aleurone layers incubated with-out GA₃ only three peaks of activity were found. In incubation media, four isoenzymes were found after GA₃ treatment and two were found after incubation without GA₃. We conclude that at least five -amylase isoenzymes are synthesized by the ER of barley aleurone layers and that this membrane system is involved in the sequestration and transport of four of these isoenzymes.Abbreviations CHA cyclohepataamylose - DEAE-cellulose diethylaminoethyl-cellulose - ER endoplasmic reticulum - GA₃ gibberellic acid - SDS-PAGE sodium-dodecyl-sulfate polyacrylamide-gel electrophoresis     

Structural properties of phospholipids in the rat liver inner mitochondrial membrane. A P-NMR study

1. 1. The ³¹P-NMR characteristics of intact rat liver mitochondria, mitoplasts and isolated inner mitochondrial membranes, as well as mitochondrial phosphatidylethanolamine and phosphatidylcholine, have been examined.

2. 2. Rat liver mitochondrial phosphatidylethanolamine hydrated in excess aqueous buffer undergoes a bilayer-to-hexagonal (H_II) polymorphic phase transition as the temperature is increased through 10°C, and thus prefers the H_II) arrangement at 37°C. Rat liver mitochondrial phosphatidylcholine, on the other hand, adopts the bilayer phase at 37°C.

3. 3. Total inner mitochondrial membrane lipids, dispersed in an excess of aqueous buffer, exhibit ³¹P-NMR spectra consistent with a bilayer arrangement for the majority of the endogeneous phospholipids; the remainder exhibit spectra consistent with structure allowing isotropic motional averaging. Addition of Ca²⁺ results in hexagonal (H_II) phase formation for a portion of the phospholipids, as well as formation of ‘lipidic particles’ as detected by freeze-fracture techniques.

4. 4. Preparations of inner mitochondrial membrane at 4 and 37°C exhibit ³¹P-NMR spectra consistent with a bilayer arrangement of the large majority of the endogenous phospholipids which are detected. Approx. 10% of the signal intensity has characteristics indicating isotropic motional averaging processes. Addition of Ca²⁺ results in an increase in the size of this component, which can become the dominant spectral feature.

5. 5. Intact mitochondria, at 4°C, exhibit ³¹P-NMR spectra arising from both phospholipid and small water-soluble molecules (ADP, P_i, etc.). The phospholipid spectrum is characteristic of a bilayer arrangement. At 37°C the phospholipids again give spectra consistent with a bilayer; however, the labile nature of these systems is reflected by increased isotropic motion at longer (at least 30 min) incubation times.

6. 6. It is suggested that the uncoupling action of high Ca²⁺ concentrations on intact mitochondria may be related to a Ca²⁺-induced disruption of the integrity of the inner mitochondrial phospholipid bilayer. Further, the possibility that non-bilayer lipid structures such as inverted micelles occur in the inner mitochondrial membrane cannot be excluded.

24-methylenepollinastanol and 24-dehydropollinastanol—two sterols of pollen

The esterified and unesterified sterol fractions of bee-gathered mixed pollens were examined, and total sterol composition was determined. Two new sterols of pollens, 14α-methyl-9β,19-cyclo-5α-cholest-24-en-3β-ol (24-dehydropollinastanol) and 14α-methyl-5α-ergost-24(28)-en-3β-ol (24-methylenepollinastanol) were isolated and identified. Both sterols were found primarily in the esterified sterol fraction, and 24-methylenepollinastanol accounted for 43% of the sterols of this fraction. 24-Dehydropollinastanol and four other sterols which also contain a 9β,19-cyclopropane ring were found only in the esterified sterol fraction. 24-Methylenecholesterol was the major sterol of the unesterified sterol fraction.     

A P-NMR saturation transfer study of the regulation of creatine kinase in the rat heart

(1) ³¹P nuclear magnetic resonance was used to measure the creatine kinase-catalysed fluxes in Langendorff-perfused rat hearts consuming oxygen at different rates and using either of two exogenous substrates (11 mM glucose or 5 mM acetate). (2) Fluxes in the direction of ATP synthesis were between 3.5–12-times the steady-state rates of ATP utilization (estimated from rates of O₂-consumption), demonstrating that the reaction is sufficiently rapid to maintain the cytosolic reactants near their equilibrium concentrations. (3) Under all conditions studied, the cytosolic free [ADP] was primarily responsible for regulating the creatine kinase fluxes. The enzyme displayed a K_m for cytosolic ADP of 35 μM and an apparent V_max of 5.5 mM/s in the intact tissue. (4) Although the reaction is maintained in an overall steady-state, the measured ratio of the forward flux (ATP synthesis) to the reverse flux (phosphocreatine synthesis) was significantly greater than unity under some conditions. It is proposed that this discrepancy may be a consequence of participation of ATP in reactions other than the PCr /ag ATP or ATP /ag ADP + P_i interconversions specifically considered in the analysis. (5) The results support the view that creatine kinase functions primarily to maintain low cytosolic concentrations of ADP during transient periods in which energy utilization exceeds production.     

Lipid-polyethylene glycol interactions: II. Formation of defects in bilayers

Summary Polyethylene glycol, a known cell fusogen, is found to induce the formation of structural defects in egg phosphatidylcholine multilamellar vesicles, as shown by freeze-fracture microscopy.³¹P NMR spectra of these vesicles reveal the existence of a nonbilayer (isotropic) phase. The observed disruption in the bilayers is believed to be associated with an intermediate stage of membrane fusion.Abbreviations PEG Polyethylene glycol - IMP Intramembranous particle - PC Phosphatidylcholine - PS Phosphatidylserine - SUV Small unilamellar vesicles - MLV Multilamellar vesicles - DPPC Dipalmitoyl phosphatidylcholine - DSC Differential scanning calorimetry - DMPC Dimyristoylphosphatidylcholine - T _c Phase transition temperature     

24β-Ethyl-31-norlanosta-8,25(27)-dien-3β-ol and 24β-ethyl-25(27)-dehydrolophenol in seeds of three cucurbitaceae species

The structures of two 4α-methylsterols is isolated from Cucumis sativus(Cucurbitaceae) seeds were determined based mainly on their ¹³CNMR spectra as 24β-ethyl-31-norlanosta-8,25(27)-dien-3β-ol and 24β-ethyl-25(27)- dehydrolophenol, respectively, of which the former is a new sterol from natural sources. These two 4α-methylsterols were identified in the seeds of two other Cucurbitaceae species, Lagenaria leucantha var. Gourda and Citrullus battich. The probable biogenetic significance of the two 4α-methylsterols is discussed. Other 4α-methylsterols identified in the seeds of the three Cucurbitaceae species were obtusifoliol, cycloeucalenol and gramisterol.     

P nuclear magnetic resonance spectroscopy of lipopolysaccharides from pseudomonas aeruginosa

Intact lipopolysaccharide antigens isolated from seven different immunotypes of Pseudomonas aeruginosa have been examined by ³¹P-NMR spectroscopy. These macromolecular complexes contain phosphorus covalently attached to the carbohydrate residues present in the lipid A moiety and the ‘core’ oligosaccharide region. The spectral signals for various ortho- and pyro-phosphoric esters were observed. All phosphate groups appeared to be mono-esterified. Certain shifts characteristic for phosphate diester groups, observed in lipopolysaccharide complexes from other Gram-negative bacteria, were absent. Furthermore, no evidence was found to indicate that phosphate groups are involved in the covalent linkage of individual lipopolysaccharide complexes to form dimers or trimers.     

Optimization of Insecticidal Triterpene Derivatives by Biomimetic Oxidations with Hydrogen Peroxide and Iodosobenzene Catalyzed by MnIII and FeIII Porphyrin Complexes

Semisynthetic functionalized triterpenes (4α,14‐dimethyl‐5α,8α‐8,9‐epoxycholestan‐3β‐yl acetate; 4α,14‐dimethyl‐5α‐cholest‐8‐ene‐3,7,11‐trione; 4α,14‐dimethyl‐5α‐cholesta‐7,9(11)‐dien‐3‐one and 4α,14‐dimethyl‐5α‐cholest‐8‐en‐3β‐yl acetate), previously prepared from 31‐norlanostenol, a natural insecticide isolated from the latex of Euphorbia officinarum, have been subjected to oxidation with hydrogen peroxide (H₂O₂) and iodosobenzene (PhIO) catalyzed by porphyrin complexes (cytochrome P‐450 models) in order to obtain optimized derivatives with high regioselectivity. The main transformations were epoxidation of the double bonds and hydroxylations of non‐activated C–H groups and the reaction products were 25‐hydroxy‐4α,14‐dimethyl‐5α‐cholesta‐7,9(11)‐dien‐3β‐yl acetate (59 %), 25‐hydroxy‐4α,14‐dimethyl‐5α‐cholest‐8‐ene‐3,7,11‐trione (60 %), 4α,14‐dimethyl‐5α,7β‐7,8‐epoxycholest‐9(11)‐en‐3‐one (22 %), 8‐hydroxy‐4α,14‐dimethyl‐5α‐cholest‐9(11)‐ene‐3,7‐dione (16 %), 12α‐hydroxy‐4α,14‐dimethyl‐5α,7β‐7,8‐epoxycholest‐9(11)‐en‐3‐one (16 %), and 4α,14‐dimethyl‐5α,8α‐8,9‐epoxycholestan‐3β‐yl acetate (26 %), respectively. We also investigated the insect (Myzus persicae, Rhopalosiphum padi and Spodoptera littoralis) antifeedant and postingestive effects of these terpenoid derivatives. None of the compounds tested had significant antifeedant effects, however, all were more effective postingestive toxicants on S. littoralis larvae than the natural compound 31‐norlanostenol, with 4α,14‐dimethyl‐5α,8α‐8,9‐epoxycholestan‐3β‐yl acetate being the most active. The study of their structure–activity relationships points out at the importance of C3 and C7 substituents.     

Proteomic signatures of in vivo muscle oxidative capacity in healthy adults

Adequate support of energy for biological activities and during fluctuation of energetic demand is crucial for healthy aging; however, mechanisms for energy decline as well as compensatory mechanisms that counteract such decline remain unclear. We conducted a discovery proteomic study of skeletal muscle in 57 healthy adults (22 women and 35 men; aged 23–87 years) to identify proteins overrepresented and underrepresented with better muscle oxidative capacity, a robust measure of in vivo mitochondrial function, independent of age, sex, and physical activity. Muscle oxidative capacity was assessed by ³¹P magnetic resonance spectroscopy postexercise phosphocreatine (PCr) recovery time (τ_PCr) in the vastus lateralis muscle, with smaller τ_PCr values reflecting better oxidative capacity. Of the 4,300 proteins quantified by LC‐MS in muscle biopsies, 253 were significantly overrepresented with better muscle oxidative capacity. Enrichment analysis revealed three major protein clusters: (a) proteins involved in key energetic mitochondrial functions especially complex I of the electron transport chain, tricarboxylic acid (TCA) cycle, fatty acid oxidation, and mitochondrial ABC transporters; (b) spliceosome proteins that regulate mRNA alternative splicing machinery, and (c) proteins involved in translation within mitochondria. Our findings suggest that alternative splicing and mechanisms that modulate mitochondrial protein synthesis are central features of the molecular mechanisms aimed at maintaining mitochondrial function in the face of impairment. Whether these mechanisms are compensatory attempt to counteract the effect of aging on mitochondrial function should be further tested in longitudinal studies.