State of aggregation of the (Ca2+ + Mg2+)-ATPase studied using saturation-transfer electron spin resonance

The state of aggregation of the (Ca2+ + Mg2+)-ATPase in the membrane of sarcoplasmic reticulum and in reconstituted membrane systems has been studied using saturation-transfer electron spin resonance (ST-ESR). Saturation-transfer ESR spectra show that in the sarcoplasmic reticulum, the ATPase is relatively free to rotate, with an effective rotational correlation time of approx. 33 microseconds at 4 degrees C, consistent with a monomeric or dimeric structure. The rate of rotation is observed to decrease with decreasing molar ratio of lipid to protein. In reconstituted systems, rotational motion of the ATPase on the millisecond time scale ceases when the lipids are in the gel phase. Addition of decavanadate, which causes the formation of crystalline arrays in negatively stained electron micrographs, results in only a small reduction in rotation rate for the ATPase in the membrane. The experiments are interpreted in terms of a short-lived (on the millisecond time scale) protein-protein interaction, with the formation of crystalline clusters of ATPase molecules which form and melt rapidly.     

Effects of bilayer thickness on the activity of diacylglycerol kinase of Escherichia coli.

We have developed a procedure for the reconstitution of Escherichia coli diacylglycerol kinase (DGK) into phospholipid bilayers containing diacylglycerol substrate. When DGK is reconstituted into a series of phosphatidylcholines containing monounsaturated fatty acyl chains, activity against dihexanoylglycerol (DHG) as a substrate was found to be markedly dependent on the fatty acyl chain length with the highest activity in dioleoylphosphatidylcholine [di(C18:1)PC] and a lower activity in bilayers with shorter or longer fatty acyl chains. Low activities in the short chain phospholipid dimyristoleoylphosphatidylcholine [di(C14:1)PC] followed from an increase in the K(m) value for DHG and ATP, with no effect on v(max). In contrast, in the long chain lipid dierucoylphosphatidylcholine [di(C24:1)PC], the low activity followed from a decrease in v(max) with no effect on K(m). In mixtures of two phosphatidylcholines with different chain lengths, the activity corresponded to that expected for the average chain length of the mixture. Cholesterol increased the activity in di(C14:1)PC but slightly decreased it in di(C18:1)PC or di(C24:1)PC, effects that could follow from changes in bilayer thickness caused by cholesterol.     

Evolutionary conservation of the chromosomal configuration and regulation of amylase genes among eight species of the Drosophila melanogaster species subgroup

Nuclear DNA was extracted from each of the eight species comprising the Drosophila melanogaster species subgroup. Southern hybridization of this DNA by using a molecular probe specific for the alpha-amylase coding region showed that the duplicated structure of the amylase locus, first found in D. melanogaster, is conserved among all species of the melanogaster subgroup. Evidence is also presented for the concerted evolution of the duplicated genes within each species. In addition, it is shown that the glucose repression of amylase gene expression, which has been extensively studied in D. melanogaster, is not confined to this species but occurs in all eight members of the species subgroup. Thus, both the duplicated gene structure and the glucose repression of Drosophila amylase gene activity are stable over extended periods of evolutionary time.      

Surface-active phospholipase A2 in mouse spermatozoa

The partial characterization of a calcium-dependent phospholipase A2 associated with membranes of mouse sperm is described. Intact and sonicated sperm had comparable phospholipase A2 activity which was maximal at pH 8.0 using [1-14C]oleate-labeled autoclaved Escherichia coli or 1-[1-14C]stearoyl-2-acyl-3-sn-glycerophosphorylethanolamine as substrates. More than 90% of the activity was sedimented when the sperm sonicate was centrifuged at 100 000 X g, indicating that the enzyme is almost totally membrane-associated. The activity is stimulated 200% during the ionophore-induced acrosome reaction and is almost equally distributed between plasma/outer acrosomal and inner acrosomal membrane fractions. The membrane-associated phospholipase A2 had an absolute requirement for low concentrations of Ca2+; Sr2+, Mg2+ and other divalent and monovalent cations would not substitute for Ca2+. In the presence of optimal Ca2+, zinc and gold ions inhibited the activity while Cu2+ and Cd2+ were without effect. Incubation of sperm sonicates with 1-[1-14C]stearoyl-2-acyl-3-sn-glycerophosphorylethanolamine in the presence and absence of sodium deoxycholate demonstrated the presence of phospholipase A2 and lysophospholipase activities. No phospholipase A1 activity was detectable. Indomethacin, sodium meclofenamate and mepacrine, but not dexamethasone or aspirin, inhibited the sperm phospholipase A2 activity. Preincubation with p-bromophenacyl bromide inhibited phospholipase A2, suggesting the presence of histidine at the active site. The enzyme may play an important role in the membrane fusion events in fertilization.     

Interaction of fatty acids with lipid bilayers

We present a method by which it is possible to describe the binding of fatty acids to phospholipid bilayers. Binding constants for oleic acid and a number of fatty acids used as spectroscopic probes are deduced from electrophoresis measurements. There is a large shift in $\text{p}\text{K}$ value for the fatty acids on binding to the phospholipid bilayers, consistent with stronger binding of the uncharged form of the fatty acid. For dansylundecanoic acid, fluorescence titrations are consistent with the binding constants derived from the electrophoresis experiments. For 12-(9-anthroyloxy)stearic acid, fluorescence and electrophoresis data are inconsistent, and we attribute this to quenching of fluorescence at high molar ratios of 12-anthroylstearic acid to phospholipid in the bilayer.     

Growth of Phytomonas serpens in a Defined Medium; Nutritional Requirements

ABSTRACT. Three strains of Phytomonas serpens two from tomatoes, Lycopersicon esculentum one from the insect Phtia picta (Hemiptera, Coreidae), were cultivated in a chemically defined medium developed from a defined medium for cultivating insect flagellates. Besides organic growth factors required by other insect trypanosomatids this flagellate requires, serine and inositol. Glutamine stimulates growth, and, surprisingly, does not require heme.     

Phytoplankton and periphyton responses to in situ experimental nutrient enrichment in a shallow subtropical lake

Experiments were performed in situ in shallow, subtropical LakeOkeechobee (Florida. USA) to quantify and compare the responsesof phytoplanklon (in 20 I clear polycarbonate carboys) and periphyton(on nutrient-diffusing clay substrates) to additions of nitrogenand/or phosphorus. During early and late summer. 1994, bothassemblages were nitrogen limited or co-limited by nitrogenand phosphorus, indicating the potential for competition betweenbenthic and planktonic communities. During late summer, therewas evidence that high phytoplankton biomass reduced light penetrationthrough the water column and may have suppressed periphytongrowth. The similar phytoplankton and periphyton taxonomic structures,both dominated by Lyngbya sp. and pennate diatoms, suggestedthat in shallow regions of this lake, resuspended meroplanktonmight account for a large portion of phytoplankton biomass.This phenomenon has been observed in other shallow, wind-drivenFlorida lakes.     

Localisation of sulfakinin neuronal pathways in the blowfly Calliphora vomitoria

The distribution of neurones immunoreactive to antisera raised against the undecapeptide C-terminal fragment of drosulfakinin II (DrmSKII), Asp-Gln-Phe-Asp-Asp-Tyr(SO₃H)-Gly-His-Met-Arg-Phe-NH₂, has been studied in the blowfly Calliphora vomitoria. Antisera were preabsorbed with combinations of the parent antigen, the tetrapeptide Phe-Met-Arg-Phe-NH₂ and cholecystokinin, the vertebrate sulfated octapeptide (CCK-8), Asp-Tyr(SO₃H)-Met-Gly-Trp-Met-Asp-Phe-NH₂, in order to ensure specificity for the sulfakinin peptides of C. vomitoria (the nonapeptide callisulfakinin I is identical to drosulfakinin I and callisulfakinin II differs from DrmSK II only by the presence of -Glu³-Glu⁴- in place of -Asp³-Asp⁴-). Only four pairs of sulfakinin-immunoreactive neurones have been visualised in the entire nervous system. These occur in the brain: two pairs of cells situated medially in the caudo-dorsal region close to the roots of the ocellar nerve and two other pairs at the same level but positioned more laterally. Despite the small number of sulfakinin-immunoreactive cells, there are extensive projections to many areas of neuropile in the brain and the thoracic ganglion. The pathway of the medial sulfakinin cells extends into each of the three thoracic ganglia and a metameric arrangement of sulfakinin neuronal projections is also seen in the abdominal ganglia. Neither the dorsal neural sheath of the thoracic ganglion, nor the abdominal nerves contain sulfakinin-immunoreactive material. These observations suggest that the sulfakinins of the blowfly function as neurotransmitters or neuromodulators. They do not appear to have a direct role in gut physiology, as has been shown by in vitro bioassays for the sulfakinins of orthopterans and blattodeans. In addition to the neurones that display specific sulfakinin immunoreactivity, other cells within the brain and thoracic ganglion are immunoreactive to cholecystokinin/gastrin antisera. There are, therefore, at least two types of dipteran neuropeptides with amino acid sequences that are similar to the vertebrate molecules cholecystokinin and gastrin.