Radiation inactivation of membrane proteins: Molecular weight estimates in situ and after Triton X-100 solubilization

Target size analysis by radiation inactivation is widely used for molecular weight determination of membrane enzymes and receptors in situ without the need for prior solubilization or purification. However, since most molecular weight data available in the literature on membrane proteins involve the use of detergents for solubilization, the target sizes of membrane proteins in situ and after solubilization by detergent treatment have been compared. Using data from the literature and personal results, three different types of behavior of membrane proteins in presence of detergents were found: (i) uncoupling of subunits (electric eel acetylcholinesterase, placental steroid sulfatase, and human nonspecific β-glucosidase); (ii) coupling of protein molecules (mouse liver neuraminidase, and rat liver insulin receptor regulatory component); and (iii) no major change in quaternary structure (rat liver insulin receptor, kidney γ-glutamyltransferase, asialoglycoprotein receptor, insulin degrading enzyme, and human leucocyte neuraminidase). For all these proteins, there is a statistically significant increase in target size of about 24% over the value obtained in situ without detergent. A relatively large body of literature data involving a variety of membrane proteins, membrane types, and irradiation conditions (electron accelerators or ⁶⁰Co sources, and proteins irradiated in lyophilized form or frozen solution) was examined, and it was concluded that target sizes of membrane proteins, irradiated in the presence of Triton X-100, should be diminished by a factor of about 24% to obtain the molecular weight value.     

Ultrastructure of the Somatic and Buccal Cortex of the Tetrahymenine Hymenostome Glaucoma chattoni

SYNOPSIS The membranes, epiplasm, and fiber systems are described in the somatic cortex of Glaucoma chattoni strain HZ-1. Kinetodesmal fibers, postciliary and transverse microtubular ribbons, basal microtubules, transverse fibers and transverse accessory material are associated with kinetosomes. Longitudinal microtubular ribbons and mitochondria occur interkinetally. In the buccal cortex, the membranes, epiplasm and fibers of the 3 membranelles, the paroral kinety, the ribbed wall, and the cytostome are described. Comparisons between G. chattoni and other ciliates reveal ultrastructural differences of possible systematic significance. In the somatic cortex of this and other tetrahymenines. Iongitudinal microtubular ribbons and basal microtubules occur concurrently. In the buccal cortex, alveoli are absent in tetrahymenine membranelles. A table is presented of the fiber systems associated with single somatic kinetosomes of various ciliates whose cortical ultrastructure has been studied to date.     

Potassium-induced insulin release and voltage noise measurements in single mouse islets of Langerhans

Summary Insulin release and membrane potential fluctuations in response to increased extracellular potassium [K⁺] _o have been measured in single perifused islets of Langerhans from normal mice. An increase in [K⁺] _o from 5mm to 50mm induced a transient insulin release with a peak at about 1 min. The peak value was [K⁺] _o -dependent but the half-timet _1/2 for the decline was constant at nearly 1 min. 2.5mm cobalt completely inhibited the potassium-induced stimulation of insulin release. The insulin release elicited by 28 and 50mm [K⁺] _o was similar in terms of peak, total release and half-time from maximum release. Stepwise increase in [K⁺] _o from 10 to 28 to 50mm resulted in a normal response to 28mm but no peak of release after the 28 to 50mm increase. The results indicate good correlation between excess voltage noise, thought to reflect calcium channel activity, and insulin release evoked by changing extracellular potassium.     

Structural changes in (Na+ + K+)-ATPase accompanying detergent inactivation

Structural changes in the purified $\text{(}\text{Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ accompanying detergent inactivation were investigated by monitoring changes in light scattering, intrinsic protein fluorescence, and tryptophan to β-parinaric acid fluorescence resonance energy transfer. Two phases of inactivation were observed using the non-ionic detergents, digitonin, Lubrol WX and Triton X-100. The rapid phase involves detergent monomer insertion but little change in protein structure or little displacement of closely associated lipids as judged by intrinsic protein fluorescence and fluorescence resonance energy transfer. Lubrol WX and Triton X-100 also caused membrane fragmentation during the rapid phase. The slower phase of inactivation results in a completely inactive enzyme in a particle of 400 000 daltons with 20 mol/mol of associated phospholipid. Fluorescence changes during the course of the slow phase indicate some dissociation of protein-associated lipids and an accompanying protein conformational change. It is concluded that non-parallel inhibition of $\text{(}\text{Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ and $\text{p-}\text{nitrophenylphosphate}$ activity by digitonin (which occurs during the rapid phase of inactivation) is unlikely to require a change in the oligomeric state of the enzyme. It is also concluded that at least 20 mol/mol of tightly associated lipid are necessary for either $\text{(}\text{Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ or $\text{p-}\text{nitrophenylphosphatase}$ activity and that the rate-limiting step in the slow inactivation phase involves dissociation of an essential lipid.     

Non-random X-chromosome expression early in mouse development

We have used a sensitive electrophoretic technique for estimating the activity, or ratio, of two allozymes of the X-chromosome-linked enzyme phosphoglycerate kinase (PGK-1), in order to investigate the randomness of X-chromosome expression in the derivatives of the three primary cell lineages of the early mouse conceptus. The maternally derived Pgk-1 allele is preferentially expressed in the derivatives of the primitive endoderm and trophectoderm lineages at 6 1/2 days post coitum in Pgk-1^a/Pgk-1^b heterozygous conceptuses, and in the one informative 5 1/2-day heterozygous conceptus analysed. This evidence for preferential expression of the maternally derived X chromosome (X^m), so soon after the time of X-chromosome inactivation, favors the possibility that the preferential expression of X^m is a consequence of primary non-random X-chromosome inactivation, rather than a secondary selection phenomenon. The majority of embryos analysed at 4 1/2 and 5 1/2 days pc produced only a single PGK-1 band, corresponding to the allozyme produced by the Pgk-1 allele on X^m, although 50% of these embryos should have been heterozygous females. Possible explanations are discussed.     

Molecules and Morphologies: The Perpetuation of Pattern in the Ciliated Protozoa*†

SYNOPSIS. Three apparently conflicting generalizations concerning the relationships between molecules and cell structure may be derived from studies on cellular patterning in the ciliates. (A) Cells with identical genes and molecular composition may have different hereditary patterns. (B) Genes, through their prescribed molecular derivatives, constrain the modes of pattern permutation and define the states of greatest stability. (C) Cells with identical hereditary patterns may have entirely different genes and molecular compositions. These principles may be reconciled through the recognition that they are characteristically applicable over different time intervals. Hereditary differences within a clone and without related molecular differences (principle A) may persist for hundreds of cell generations, but they are resolved eventually within a constant environment (principle B) as the configuration of minimal free energy is approached. On an even longer time scale, molecular substitutions have occurred for many or most components of the cell, but these have been constrained by selective pressures on an ancient design (principle C) that disallow substitutions affecting certain form-function relations which have been elevated to an adaptive peak.     

Properties of immobilized β-d-galactosidase from Bacillus circulans

Partially purified β-d-galactosidase (β-d-galactoside galactohydrolase, EC 3.2.1.23) from Bacillus circulans showed high activity towards both pure lactose and lactose in skim milk, and a better thermal stability than the enzyme from yeast or Escherichia coli. During the course of hydrolysis of lactose catalysed by the enzyme, considerable amounts of oligosaccharides were produced. β-d-Galactosidase from B. circulans was immobilized onto Duolite ES-762, Dowex MWA-1 and sintered alumina by adsorption with glutaraldehyde treatment. The highest activity for hydrolysis of lactose was obtained with immobilization onto Duolite ES-762. During a continuous hydrolysis of lactose, the immobilized enzyme was reversibly inactivated, probably due to oligosaccharides accumulating in the gel. The inactivation was reduced when a continuous reaction was operated at a high percent conversion of lactose in a continuous stirred tank reactor (CSTR). The half-life of the immobilized enzyme was estimated to be 50 and 15 days at 50 and 55°C, respectively, when the reaction was carried out in a CSTR with a percent conversion of lactose >70%.     

Inactivation and stabilization of stabilisins in neat organic solvents

The stability of the serine proteases from Bacillus amyloliquefaciens (subtillisin BPN') and Bacillus licheniformis (subtilisin Carlsberg) was investigated in various anhydrous solvents at 45 degrees C. The half-life of subtilisin BPN' in dimethyl-formamide dramatically depends on the pH of the aqueous solutions from which the enzyme was lyophilized, increasing from 48 min to 20 h when the pH is raised from 6.0 to 7.9. Both subtilisins exhibited substantial inactivation during multihour incubations in tert-amyl alcohol and acetonitrile when enzymatic activities were also measured in these solvents; however, when the enzymes were assayed in water instead, hardly any loss of activity was detected. This surprising difference appears to stem from the partitioning of the bound water essential for catalytic activity from the enzymes into the solvents. When assayed in organic solvents, this time-dependent stripping of water results in decay of enzymatic activity; however, when assayed in water, where the dehydrated subtilisins can undergo rehydration thereby recovering catalytic activity, little inactivation is observed. In agreement with this hypothesis, the addition of small quantities of water tert-amyl alcohol stabilized the subtilisins in it even when enzymatic activity was measured in the nonaqueous solvent. Ester substrates (vinyl butyrate and trichloroethyl butyrate) greatly enhanced the stability of both subtilisins in organic solvents possibly because of the formation of the acyl-enzymes.     

Inactivation kinetics and pharmacology distinguish two calcium currents in mouse pancreatic B-cells

Summary Voltage-dependent calcium currents were studied in cultured adult mouse pancreatic B-cells using the whole-cell voltage-clamp technique. When calcium currents were elicited with 10-sec depolarizing command pulses, the time course of inactivation was well fit by the sum of two exponentials. The more rapidlyinactivating component had a time constant of 75±5 msec at 0 mV and displayed both calcium influx- and voltage-dependent inactivation, while the more slowly-inanctivating component had a time constant of 2750±280 msec at 0 mV and inactivated primarily via voltage. The fast component was subject to greater steady-state inactivation at holding potentials between –100 and –40 mV and activated at a lower voltage threshold. This component was also significantly reduced by nimodipine (0.5 m) when a holding potential of –100 mV was used, whereas the slow component was unaffected. In contrast, the slow component was greatly increased by replacing external calcium with barium, while the fast component was unchanged. Cadmium (1–10 m) displayed a voltage-dependent block of calcium currents consistent with a greater effect on the high-threshold, more-slowly inactivating component. Taken together, the data suggest that cultured mouse B-cells, as with other insulin-secreting cells we have studied, possess at least two distinct calcium currents. The physiological significance of two calcium currents having distinct kinetic and steady-state inactivation characteristics for B-cell burst firing and insulin secretion is discussed.