Febs Letters

doi: 10.1016/0014-5793(86)81072-9pmid: N/A

Timmerman, M.P.; Ashley, C.C.

doi: 10.1016/0014-5793(86)81073-0pmid: 3803567

Two questions bearing on the use of fura‐2 to measure transient changes in intracellular Ca2+ concentration have been addressed. To investigate fura‐2 intracellular binding, the amounts of fura‐2 and [14C]glycine in Balanus nubilus myofibrillar bundles after loading were determined and their intracellular apparent diffusion constants measured. No significant fura‐2 immobilisation occurs under the conditions used. The apparent diffusion constant for fura‐2 in aqueous solution was determined. The relationship between half‐time for relaxation of force and fura‐2 fluorescence transients, and intracellular fura‐2 concentration, in voltageclamped single muscle fibres was examined. Significant buffering of the Ca2+ transient occurred at fura‐2 concentrations above ~ 6 μM.

Cawston, Timothy E.; Mercer, Elizabeth

doi: 10.1016/0014-5793(86)81074-2pmid: 2433154

The binding of collagenase to both α2‐niacroglobulin and the tissue inhibitor of metalloproteinases was studied using purified materials. Collagenase bound preferentially to α2‐macroglobulin although no transfer of collagenase to α2‐macroglobulin occurred if the enzyme was first mixed with the tissue inhibitor of metalloproteinases. The sequences of amino acids in both inhibitors likely to be responsible for the binding of collagenase are discussed and compared to the cleavage site in the collagen molecule.

Dodd, I.; Jalalpour, S.; Southwick, W.; Newsome, P.; Browne, M.J.; Robinson, J.H.

doi: 10.1016/0014-5793(86)81075-4pmid: 3100325

Recombinant tissue‐type plasminogen activator (rt‐PA) from cultures of a genetically manipulated Bowes melanoma cell line (TRBM6) was purified in batches of average volume 451 using an autoclavable, reusable, continuous chromatography system comprising zinc chelate‐Sepharose CL4B and lysine‐Sepharose CL4B. After eight successive purifications the rt‐PA was ultrafiltered to yield a preparation containing 4.9 mg protein/ml and 2.7 × 106 . Analysis by SDS‐polyacrylamide gel electrophoresis followed by staining with Coomassie brilliant blue R250 showed major protein bands at M r = 63000 and 65000; most of the material was in the 1‐chain form. The potential usefulness of a simple, rapid continuous chromatography system that can be operated under aseptic conditions is discussed.

Ho, Chewn-Lang; Ko, Jane-Ling

doi: 10.1016/0014-5793(86)81076-6pmid: 3803573

By gel permeation on a Fractogel TSK HW 50 column followed by ion‐exchange chromatography on carboxymethylcellulose CM 52, a lethal protein, designated hornetin, was purified from the venom of Vespa flavitarsus. Hornetin is a highly basic protein (pI 10.2) with a molecular mass of about 32 kDa. Its amino acid composition is characterized by a high content of lysine, aspartic and glutamic acid, and is devoid of tryptophan and cysteine. The lack of cysteine in the molecule is distinct from other known vespid venom proteins of comparable size. The i.v. LD50 of the toxin is 0.42 μg per g mouse. Assayed on the red blood cells of the mouse and guinea‐pig as well as isolated nerve muscle preparations of the chick and mouse, hornetin showed direct hemolytic activity and presynaptic neurotoxicity at microgram level and displayed musculotropic effect at higher concentrations.

Usanga, E.A.; O'Brien, Estella; Luzzato, L.

doi: 10.1016/0014-5793(86)81077-8pmid: 3542560

We report that the mitotic inhibitor, vinblastin (VLB), is highly toxic to the malarial parasite, Plasmodium falciparum. In cultures in vitro growth is inhibited by 50% at a VLB level of about 28 nM, and totally abolished at a level of 100 nM. By tests on synchronized cultures we have found that the effect of VLB takes place at the trophozoite stage. Colcemid also inhibits schizogony with somewhat different kinetics. By mutagenesis with nitrosoguanidine followed by VLB selection we have isolated a VLB‐resistant mutant which exhibits cross‐resistance to vincristine. These data suggest a critical role of microtubules in the asexual schizogonic cycle of P. falciparum.

Sundblad, Lars-Göran; Palmqvist, Kristin; Samuelsson, Göran

doi: 10.1016/0014-5793(86)81078-Xpmid: N/A

Luminescence decay kinetics was shown to be polyphasic with two relative maxima observed, when high CO2 adapted cells of Scenedesmus obliquus were stressed by CO2 deficiency. Low CO2 adapted cells exhibited a single maximum. Luminescence from high CO2 adapted cells under high CO2 conditions decayed asymptotically without maxima. The magnitude of the transthylakoid ΔpH was shown to be related to the intensity of luminescence in an antiparallel way. The relaxation kinetics of the ‐ΔpH was shown to be complex, with an interval of increasing or maintained ΔpH. A working hypothesis is presented in order to explain polyphasic luminescence decay kinetics in terms of luminescence quenching by the transthylakoid ΔpH.

Nicolay, Klaas; Smaal, Erik B.; de Kruijff, Ben

doi: 10.1016/0014-5793(86)81079-1pmid: 3803574

2H NMR has been used to probe the effects of ethylene glycol at the level of the acyl chains in liposomes prepared from dioleoylphosphatidic acid or dioleoylphosphatidylcholine, labeled with 2H at the 11‐position of both oleic acid chains. Increasing concentrations of ethylene glycol lead to a proportional and substantial decrease in the quadrupolar splittings, measured from the 2H NMR spectra of both liposomal systems, indicative of acyl chain disordering.

Breton, J.; Martin, J.-L.; Petrich, J.; Migus, A.; Antonetti, A.

doi: 10.1016/0014-5793(86)81080-8pmid: N/A

Reaction centers from the photosynthetic bacterium Rhodopseudomonas sphaeroides have been excited either in the bacteriopheophytin band at 760 nm or in the accessory bacteriochlorophyll (B) band around 800 nm with laser pulses of 150 fs duration. Upon monitoring in the absorption band of the primary donor (P) at 860 nm, ultrafast energy transfer is observed which leads to the excited state of P in less than 100 fs. A transient bleaching recovering in 400 ± 100 fs is specifically detected upon excitation and observation in the 800 nm absorption band of B. However, upon direct excitation of P in the near infrared and using either normal or borohydride‐treated reaction centers, we have found no spectral or kinetic evidence indicating the presence of a transient intermediate state such as P+B−.

Dancshazy, Zsolt; Govindjee, Rajni; Nelson, Burr; Ebrey, Thomas G.

doi: 10.1016/0014-5793(86)81081-Xpmid: N/A

With increasing pH (6.0 to 10.5) there is an increasing discrepancy between the recovery rate of bacteriorhodopsin after a flash and the decay rates of the key photointermediates, Mfast and Mslow, which is not predicted by any model of the photocycle. However, a very slowly decaying absorbance change at 350 nm has kinetics which match the very slow part of the recovery of bacteriorhodopsin, suggesting a new intermediate. The difference spectra of the bacteriorhodopsin photocycle measured at pH 10.5, obtained from fitting 3 exponentials to the flash‐induced absorbance changes in the 300–560 nm wavelength range, show maxima, at 410–420 nm corresponding to the 2 forms of M and at 350 nm corresponding to the new intermediate. We propose to call this new species R350.