The synthesis of 15N- and deuterium-substituted, spin-labeled analogues of NAD+ and their use in EPR studies of dehydrogenases

Calcium efflux and EGTA-induced calcium release from an internal platelet membrane fraction have been studied after the oxalate-supported calcium uptake had reached steady state. Increasing external calcium concentrations stimulate the calcium efflux velocity, with an apparent half-maximal stimulation at about 5 microM outside calcium concentration and a maximal velocity of calcium efflux of 4.66 +/- 2.32 nmol X min-1 X mg-1. Moreover, the ratio of the liberated calcium on the loaded calcium seems to be independent of the increasing external calcium concentration. Increasing the calculated internal calcium concentration by varying the oxalate potassium concentration from 10 mM to 1 mM results in an increase of the liberated calcium from the membrane vesicles from 7.4% to 63%, respectively, without changing the calcium efflux velocity. Similar conclusions can be drawn from the observation of results from the calcium efflux and EGTA-induced calcium release methods. Moreover, calcium pump reversal does not seem to be responsible for the calcium efflux or calcium release. All these different points added to the previously described regulation of calcium efflux by the catalytic subunit of cAMP protein kinase suggest us that the mechanism of calcium liberation by the platelet membranes is different from the calcium uptake.     

Carnitine transport by rat kidney cortex slices: stimulation by dibutyryl cyclic AMP+.

The transport of carnitine by rat kidney cortex slices against a concentration gradient has been demonstrated. Similarities to other transport systems included a linear period of uptake, as well as indications of saturability of the system with increasing concentrations of substrate. The transport of carnitine was inhibited by anoxia, and carbonyl cyanide-m-chloro-phenylhydroxazone (CCC1P), an uncoupler of oxidative phosphorylation. Carnitine uptake was stimulated approximately 50% when kidney slices were treated with dibutyryl cAMP.     

On the mechanism of the chemical modification of the mitochondrial acetyl-CoA acetyltransferase by coenzyme A

The liver mitochondrial acetyl-CoA acetyltransferase (acetyl-CoA:acetyl-CoA C-acetyltransferase, EC 2.3.1.9), is involved in ketone body synthesis. The enzyme can be chemically modified and inactivated by CoASH and also by CoASH-disulfides provided glutathione is present. The unmodified enzyme shows in its denatured state 7.95 +/- 0.44 sulfhydryl groups per enzyme and in its native state 3.92 +/- 0.34 sulfhydryl groups which react with Ellmann's reagent. The modified enzyme reveals in its native state also 4.07 +/- 0.25 sulfhydryl groups per enzyme, but in its denatured state 9.10 +/- 0.51 sulfhydryl groups could be detected. Approximately four sulfhydryl groups per enzyme, unmodified or modified, can be alkylated by iodoacetamide. These results prove for each subunit the existence of two sulfhydryl groups and suggest the existence of two disulfide bridges. The CoASH modification, which should proceed at one of these disulfide groups, prevents subsequent acetylation of the enzyme and is drastically reduced in the iodoacetamide-alkylated enzyme. In the demodification of the modified enzyme, the CoASH is set free as a mixed disulfide with glutathione.     

Modulation of rat-liver mitochondrial acetyl-CoA acetyltransferase activity by a reversible chemical modification with coenzyme A

The mitochondrial acetyl-CoA acetyltransferase (acetoacetyl-CoA thiolase, EC 2.3.1.9) is involved in ketone body biosynthesis. In its unmodified state, referred to as transferase B in former publications (Huth, W. (1981) Eur. J. Biochem. 120, 557-562), the enzyme is characterized by the highest specific activity of 21.65 mumol/min per mg protein (direction of acetoacetyl-CoA synthesis); several forms of the enzyme with lower specific activities result from chemical modification by an apparent covalent binding of CoASH. The chemical modification results in an inactivation of the enzyme: a 2 h incubation with 0.2 mM CoASH at pH 8.1 at 30 degrees C inactivates up to 95%. Both processes, the CoASH-binding and the resulting inactivation, can be simultaneously reversed by treatment with glutathione. The specificity of inactivation is limited to CoASH and the intact sulfhydryl group is a prerequisite for this process. The enzyme exhibits a limited number (n = 3.2) of high-affinity (Ka = 26.7 microM) specific binding sites for CoASH. The inactivation-reactivation cycle of acetyl-CoA acetyltransferase by CoASH and glutathione may involve a protein disulfide-thiol exchange and represents a mode of control in modulating the amount of active enzyme.     

Redox conditions for stimulation of in vitro folding and assembly of the glycoprotein hormone chorionic gonadotropin

The formation of native disulfide bonds during in vitro protein folding can be limiting in obtaining biologically active proteins. Thus, optimization of redox conditions can be critical in maximizing the yield of renatured, recombinant proteins. We have employed a folding model, that of the beta subunit of human chorionic gonadotropin (hCG- beta), to investigate in vitro oxidation conditions that facilitate the folding of this protein, and have compared the in vitro rates obtained with the rate of folding that has been observed in intact cells. Two steps in the folding pathway of hCG-beta were investigated: the rate-limiting events in the folding of this protein, and the assembly of hCG-beta with, hCG-alpha. The rates of these folding events were determined with and without protein disulfide isomerase (PDI) using two different types of redox reagents: cysteamine and its oxidized equivalent, cystamine, and reduced and oxidized glutathione. Rates of the rate-limiting folding events were twofold faster in cysteamine/cystamine redox buffers than in glutathione buffers in the absence of PDI. Optimal conditions for hCG-beta folding were attained in a 2 mM glutathione buffer, pH 7.4, that contained 1 mg/mL PDI and in 10muM cysteamine/cystamine, pH 8.7, without PDI. Under these conditions, the half-time of the ratelimiting folding event was 16 to 20 min and approached the rate observed in intact cells (4 to 5 min). Moreover, folding of the beta subunit under these conditions yields a functional protein, based on its ability to assemble with the alpha subunit. The rates of assembly of hCG-beta with hCG-alpha in the cysteamine/cystamine or glutathione/PDI redox buffers were comparable (t(1/2/sb> = 9 to 12 min)). These studies show that rates of folding and assembly events that involve disulfide bond formation can be optimized by a simple buffer system composed of cysteamine and cystamine. (c) 1994 John Wiley & Sons, Inc.     

Properties of carnitine transport in rat kidney cortex slices

The properties of carnitine transport were studied in rat kidney cortex slices. Tissue: medium concentration gradients of 7.9 for L-[methyl-¹⁴C]carnitine were attained after 60-min incubation at 37°C in 40 μM substrate. L- and D-carnitine uptake showed saturability. The concentration curves appeared to consist of (1) a high-affinity component, and (2) a lower affinity site. When corrected for the latter components, the estimated K_m for L-carnitine was 90 μM and $\text{V = 22}\text{nmol/min}$ per ml intracellular fluid; for D-carnitine, $\text{K}{}_{\mathrm{<mtext>m</mtext>}}\text{= 166}\text{μM}$ and $\text{V = 15}\text{nmol/min}$ per ml intracellular fluid. The system was stereospecific for L-carnitine. The uptake of L-carnitine was inhibited by (1) D-carnitine, γ-butyrobetaine, and (2) acetyl-L-carnitine. γ-Butyrobetaine and acetyl-L-carnitine were competitive inhibitors of L-carnitine uptake. Carnitine transport was not significantly reduced by choline, betaine, lysine or γ-aminobutyric acid. Carnitine uptake was inhibited by 2,4-dinitrophenol, carbonyl cyanide $\text{m-}\text{chlorophenylhydrazone}$, N₂ atmosphere, KCN, $\text{N-}\text{ethylmaleimide}$, low temperature (4°C) and ouabain. Complete replacement of Na⁺ in the medium by Li⁺ reduced L- and D-carnitine uptake by 75 and 60%, respectively. Complete replacement of K⁺ or Ca²⁺ in the medium also significantly reduces carnitine uptake. Two roles for the carnitine transport system in kidney are proposed: (1) a renal tubule reabsorption system for the steady-state maintenance of plasma carnitine; and (2) maintenance of normal carnitine levels in kidney cells, which is required for fatty acid oxidation.     

Direct assay for creatine kinase by reversed-phase high-performance liquid chromatography

A direct assay for creatine kinase (CK) activity was developed based on the separation and quantitation of adenosine triphosphate (ATP) by high-performance liquid chromatography. The total incubation time is 13 min and the elution time for ATP is 16 min. Using lyophilized CK as the sample, a sensitivity in the range of 8 U/l (units/liter) was obtained. The method presented also has clinical significance in that CK levels in serum can easily be determined with minimal sample preparation. Using serum samples from a healthy patient and a heart attack victim, activities of 26.6 U/l and 609.0 U/l, respectively, were obtained. Because of the direct measurement of ATP, this method eliminates the coupled reactions encountered in the common spectrophotometric and colorimetric methods of analysis resulting in a simpler and inexpensive assay.     

Balbiani ring RNA in the cytoplasm of Chironomus thummi salivary gland cells

In this paper experimental results on the size, transport and stability of cytoplasmic Balbiani ring RNA and on its appearance in polysomes are presented. Cytoplasmic RNA of salivary gland cells from Chironomus thummi contains two large RNA fractions of about 20×10⁶ dalton and 10×10⁶ dalton in size. These RNA fractions correspond both to Balbiani ring BR 1 RNA and BR 2 RNA and are apparently transported from nucleus into cytoplasm without a significant size reduction. Chase experiments illustrate a great stability of giant cytoplasmic Balbiani ring RNA molecules and exclude the possibility of a precursor-product relationship between these and smaller BR RNA molecules also found in cytoplasm. A part of giant cytoplasmic Balbiani ring RNA molecules is bound to poly(U)-sepharose columns and should, therefore, contain poly(A)-sequences. — Polysomes of salivary gland cells extracted by a gentle lysis procedure and centrifuged through sucrose gradients are characterized by a rather broad sedimentation profile. Polysome sizes up to about 800 S have been detected, but in no case a distinct polysome fraction corresponding in size to Balbiani ring RNA has been observed. Hybridization of polysomal RNA with salivary gland chromosomes in situ resulted in labelling of both Balbiani rings BR 1 and BR 2.     

Early vigour in wheat: Could it lead to more severe terminal drought stress under elevated atmospheric [CO2] and semi‐arid conditions?

Early vigour in wheat is a trait that has received attention for its benefits reducing evaporation from the soil surface early in the season. However, with the growth enhancement common to crops grown under elevated atmospheric CO₂ concentrations (e[CO₂]), there is a risk that too much early growth might deplete soil water and lead to more severe terminal drought stress in environments where production relies on stored soil water content. If this is the case, the incorporation of such a trait in wheat breeding programmes might have unintended negative consequences in the future, especially in dry years. We used selected data from cultivars with proven expression of high and low early vigour from the Australian Grains Free Air CO₂ Enrichment (AGFACE) facility, and complemented this analysis with simulation results from two crop growth models which differ in the modelling of leaf area development and crop water use. Grain yield responses to e[CO₂] were lower in the high early vigour group compared to the low early vigour group, and although these differences were not significant, they were corroborated by simulation model results. However, the simulated lower response with high early vigour lines was not caused by an earlier or greater depletion of soil water under e[CO₂] and the mechanisms responsible appear to be related to an earlier saturation of the radiation intercepted. Whether this is the case in the field needs to be further investigated. In addition, there was some evidence that the timing of the drought stress during crop growth influenced the effect of e[CO₂] regardless of the early vigour trait. There is a need for FACE investigations of the value of traits for drought adaptation to be conducted under more severe drought conditions and variable timing of drought stress, a risky but necessary endeavour.