Sarcolemmal calcium binding sites in heart: II. Mathematical model for diffusion of calcium released from the sarcoplasmic reticulum into the diadic region

Summary We present a model for predicting the temporal and spatial dependence of [Ca] in the cardiac subsarcolemmal diadic region (cleft), following Ca release from the feet of the sarcoplasmic reticulum. This region is modeled as a disc 10 nm thick, 430 nm in radius, with or without Ca binding sites and open at its periphery to the cytosol. [Ca] is computed for three diffusion coefficients (100, 20 and 4% of aqueous diffusion), following release of a 20-msec square pulse sufficient to produce 50% maximal contractile force, or repetitive release (400/min) of such pulses. Numerical solutions are obtained for the general diffusion/binding problem and analytic solutions for the case of no binding sites. For the middle value of diffusion coefficient, and in the absence of binding sites, [Ca] rises to 1.5 mm in 20-msec and then falls to 0.1 m in < 3 msec. Adding binding sites reduces peak [Ca] to 0.6 mm but prolongs its decline, requiring 200 msec to reach 20 m. For repetitive release [Ca] is > 100 m for roughly half of each cycle. Two major implications of the predicted [Ca] are: (i) The effect of Ca binding sites on [Ca] will cause Ca efflux from the cleft via the NaCa exchanger (K _m (Ca) 20 m) to continue at a significant level for > 200 msec, (ii) The time constant for inactivation of release from the feet must be much greater than for activation if Cainduced Ca release is to continue for > 1–2 msec.     

Localization of 3H-dihydrotestosterone in the pituitary gland of the rhesus monkey

Summary The uptake and retention of radiolabelled dihydrotestosterone by the pituitary gland was examined in the rhesus monkey. Two animals were given an intravenous injection of 1.0g/kg ³H-dihydrotestosterone (DHT) alone while one monkey received both the labelled androgen and 100g/kg of unlabelled steroid. One and a half hours later, they were sacrificed. The pituitary glands were removed and processed for autoradiography and immunocytochemistry. Autoradiographic localization of DHT was discernible in the partes nervosa, intermedia and distalis, albeit the highest concentration of radiolabelled cells was noted in the pars distalis. Immunocytochemical staining with antibodies to rat PRL, human TSH and ovine LH revealed a population of steroid-concentrating cells that contained TSH and a second group that contained LH. None of the cells that reacted with the anti-PRL serum were radiolabelled.     

Interaction of phlorizin and sodium with the renal brush-border membraned-glucose transporter: Stoichiometry and order of binding

Summary The order and stoichiometry of the binding of phlorizin and sodium to the renal brush-border membraned-glucose transporter are studied. The experimental results are consistent with a random-binding sites is one-to-one. When the kinetics of phlorizin binding are measured as a function of increasing sodium concentration no significant variation is found in the apparent number of binding sites; however, the apparent binding constant for phlorizin decreases rapidly from approximately 16 m at [Na]=0 to 0.1 m at [Na]=100mm and approaches 0.05 m as [Na]. The experimental data are fit to a random carrier-type model of the coupled transport of sodium andd-glucose. A complete parameterization of the phlorizin binding properties of this model under sodium equilibrium conditions is given.     

The in vitro production of an anthocyanin from callus cultures of Oxalis linearis

Callus growth and the production of anthocyanins were sustained on the salts and vitamins of Murashige and Skoog. Callus growth was stimulated at a concentration of 8–32 M -naphthaleneacetic acid (NAA) and 2,4-dichlorophenoxyacetic acid (2,4-d). Benzyladenine (BA) and zeatin at 8 M inhibited callus growth whereas isopentenyladenine (iP) stimulated callus growth. NAA repressed anthocyanin production with an increase in NAA from 8–32 M. Anthocyanin synthesis was promoted by an increase in 2,4-d from 0.5 to 2 M and decreased thereafter up to a concentration 32 M 2,4-d. A concentration of 8 M BA, thidiazuron and zeatin, respectively stimulated pigment production. Sucrose stimulated callus growth at 60 mM and pigment production at 120–360 mM.Abbreviations BA 6-benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - NAA -naphthaleneacetic acid - iP isopentenyladenine - TZ thidiazuron-N-phenyl-N-1,2,3-thiadiazol-5-yl-urea - Bu-HCl Butanol-2N HCl - BAW Butanol-acetic acid-water     

Purification and characterization of flavone-specific β-glucuronidase from callus cultures of Scutellaria baicalensis Georgi

-Glucuronidase from callus cultures of Scutellaria baicalensis Georgi was purified to apparent homogeneity by fractionated ammonium-sulfate precipitation and chromatography on diethylaminoethyl-cellulose, hydroxylapatite and baicalin-conjugated Sepharose 6B. A 650-fold purification was obtained by this purification system. When subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis the purified protein migrated as a single band with a molecular mass of 55 kDa. We determined that the native enzyme has a molecular mass of 230 kDa using gel-filtration chromatography. These results suggested that the enzyme exists as a homotetramer composed of four identical 55-kDa subunits. The enzyme showed a broad pH optimum between 7.0 and 8.0. The K _m values were 9 M, 10 M, 30 M and 40 M for luteolin 3 -O--d-glucuronide, baicalin, wogonin 7-O--d-glucoronide and oroxlin 7-O--d-glucuronide, respectively. The enzyme was most active with flavone 7-O--d-glucuronides.Abbreviations BA N⁶-benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - pI isoelectric point - R _t retention time     

A combined micro-and semi-micro colorimetric determination of long-chain fatty acids from plant cutin

Summary Re-examination of the colorimetric fatty acid determination with copper nitrate, followed by complex formation with DIECA has shown that the method is not reliable if applied as described by Duncombe (1962, 1963): The Cu concentration is too high, the DIECA concentration much too low and the wavelength chosen (440 m) is suitable only for very low fatty acid concentrations.According to the results reported here the following alterations have to be adopted: The concentration of the copper nitrate solution should be 3%, a 0.5% solution of DIECA in butanol has to be used and measurements should be done at 492 m. The method described here offers the opportunity to determine fatty acid concentrations in the semi-micro range by measuring the filtered chloroform phase directly at 691 m, covering a range between 175 g/ml to 1.2 mg/ml. If the concentration turns out to be lower than 200 g F. A./ml, the same sample can be used for a micro-determination (up to 200 g/ml) at 492 m, after formation of the yellowish-brown complex by addition of 0.1 ml 0.5% butanolic DIECA solution to 1.0 ml of the chloroform phase.The method has been applied to determine the amount of free F. A. in cutin layers and cutin powder, revealing that the latter contains 5.6 times more free F. A. than the intact material. The free F. A. within the polymer seem to serve as interconnections for the main units of the cutin polylipid.     

Optimization of cell density and dilution rate in <Emphasis Type="Italic">Pichia pastoris</Emphasis> continuous fermentations for production of recombinant proteins

This paper provides an approach for optimizing the cell density (X_c) and dilution rate (D) in a chemostat for a Pichia pastoris continuous fermentation for the extracellular production of a recombinant protein, interferon (INF-). The objective was to maximize the volumetric productivity (Q, mg INF- l^–1 h^–1), which was accomplished using response surface methodology (RSM) to model the response of Q as a function of X_c and D within the ranges 150 X_c 450 g cells (wet weight) l^–1 and 0.1 _mD0.9 _m (_m=0.0678 h^–1, the maximum specific growth rate obtained from a fed-batch phase controlled with a methanol sensor). The methanol and medium feed rates that resulted in the desired X_c and D were determined based on the mass balance. From the RSM model, the optimal X_c and D were 328.9 g l^–1 and 0.0333 h^–1 for a maximum Q of 2.73 mg l^–1 h^–1. The model of specific production rate (, mg INF- g^–1 cells h^–1) was also established and showed the optimal X_c=287.7 g l^–1 and D=0.0361 h^–1 for the maximum (predicted to be 8.92×10^–3 mg^–1 g^–1 h^–1). The methanol specific consumption rate (, g methanol g^–1 cells h^–1) was calculated and shown to be independent of the cell density. The relationship between and (specific growth rate) was the same as that discovered from fed-batch fermentations of the same strain. The approach developed in this study is expected to be applicable to the optimization of continuous fermentations by other microorganisms.     

Das Cytostom von Lankesterella garnhami

Zusammenfassung Die Nahrungsaufnahme der Merozoiten von Lankesterella garnhami in den Milzzellen von Hamburger Hausspatzen (Passer d. domesticus) wurde elektronenmikroskopisch untersucht. Teile des Wirtszytoplasmas gelangen durch Abschnürung zahlreicher, etwa 20–30 m. großer Pinozytosevesikel in das Lumen der periparasitären Vakuole. Von dem Inhalt der periparasitären Vakuole werden Teile mit Hilfe mehrerer Cytostomstrukturen in Nahrungskanäle aufgenommen, von denen sich 100–200 m große Nahrungsvakuolen abschnüren, in denen die weitere Verdauung stattfindet. Das Cytostom ist durch zwei intensiv kontrastierte, konzentrische Zylinder charakterisiert, die sich von den beiden Pelliculamembranen ableiten. Der innere Durchmesser beträgt ca. 80m, der äußere Durchmesser ca. 150 m und die Tiefe im Ruhestadium mindestens 50–100 m. Die Bedeutung des Cytostoms als taxonomisches Merkmal wird diskutiert.

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The cytostome of Lankesterella garnhami

Summary The feeding mechanism of merozoites of Lankesterella garnhami in the cells of the spleen of young sparrows (Passer d. domesticus), which were captured in Hamburg, was studied by means of electron microscopy. The cytoplasm of the host cell permeates the membrane of the periparasitic vacuole by means of pinocytosis of vesicles with a diameter of 20–30 m. The parasite takes up parts of the content of the periparasitic vacuole with several cytostome structures. This structure consists of two electron dense, concentric cylinders, which originate from the two membranes of the pellicle. The cytostome is 80 m in the inner diameter and 150 m in the outer diameter. The depth of the inactive cytostomal cavity is about 50–100 m, whereas it is much prolonged in the stage of active feeding. In this stage the cytostome opens into a channel of 400–800 m and more, from which food vacuoles of 100–200 m are pinched off. Digestion occurs in the food vacuole. These findings are compared with observations of the cytostome and the micropyle in other sporozoa. Sometimes long, V-shaped invaginations of unknown signification are noticed.

     

The influence of membrane potential on chloride channels activated by GABA in rat cultured hippocampal neurons

Chloride currents were activated by a low concentration of GABA (0.5 m) in neonatal rat hippocampal neurons cultured for up to 14 days. Currents elicited by 0.5 m GABA in neurons, voltage-clamped using the whole-cell technique with pipettes containing 149 mm Cl^–, reversed close to 0 mV whether pipettes contained 144 mm Na⁺ or 140 mm Cs⁺, and were blocked by 100 m bicuculline. Current-voltage curves showed outward rectification. Single channel currents appeared in cell-attached patches when the pipette tip was perfused with pipette solution containing 0.5 m GABA and disappeared when a solution containing 100 m bicuculline plus 0.5 m GABA was injected into the pipette tip. The channels showed outward rectification and, in some patches, had a much lower probability of opening at hyperpolarized potentials. The average chord conductance in 10 patches hyperpolarized by 80 mV was 7.8±1.6 pS (sem) compared with a chord conductance of 34.1±3.5 pS (sem) in the same patches depolarized by 80 mV. Similar single channel currents were also activated in cell-free, inside-out patches in symmetrical chloride solutions when 0.5 m GABA was injected into the pipette tip. The channels showed outward rectification similar to that seen in cell-attached patches, and some channels had a lower probability of opening at hyperpolarized potentials. The average chord conductance in 13 patches hyperpolarized by 80 mV was 11.8±2.3 pS (sem) compared with 42.1±3.1 pS (sem) in the same patches depolarized by 80 mV.We are grateful to B. McLachlan and M. Robertson for their general assistance, to C. McCulloch and M. Smith for writing computer programs and to W. O'Hare for making the pipette injection device.     

Interaction among anion,cation and glucose transport proteins in the human red cell

Summary The time course of binding of the fluorescent stilbene anion exchange inhibitor, DBDS (4,4-dibenzamido-2,2-stilbene disulfonate), to band 3 can be measured by the stopped-flow method. We have previously used the reaction time constant, _DBDS, to obtain the kinetic constants for binding and, thus, to report on the conformational state of the band 3 binding site. To validate the method, we have now shown that the ID₅₀ (0.3±0.1 m) for H₂-DIDS (4,4-diisothiocyano-2,2-dihydrostilbene disulfonate) inhibition of _DBDS is virtually the same as the ID₅₀ (0.47±0.04 m) for H₂-DIDS inhibition of red cell Cl^– flux, thus relating _DBDS directly to band 3 anion exchange. The specific glucose transport inhibitor, cytochalasin B, causes significant changes in _DBDS, which can be reversed with intracellular, but not extracellular,d-glucose. ID₅₀ for cytochalasin B modulation of _DBDS is 0.1±0.2 m in good agreement withK _D =0.06±0.005 m for cytochalasin B binding to the glucose transport protein. These experiments suggest that the glucose transport protein is either adjacent to band 3, or linked to it through a mechanism, which can transmit conformational information. Ouabain (0.1 m), the specific inhibitor of red cell Na⁺,K⁺-ATPase, increases red cell Cl^– exchange flux in red cells by a factor of about two. This interaction indicates that the Na⁺,K⁺-ATPase, like the glucose transport protein, is either in contact with, or closely linked to, band 3. These results would be consistent with a transport proteincomplex, centered on band 3, and responsible for the entire transport process, not only the provision of metabolic energy, but also the actual carriage of the cations and anions themselves.     

Electron microscopic studies on the adenohypophysis of the White-Crowned sparrow,Zonotrichia leucophrys gambelii

Summary The pars distalis of the adenohypophysis of normal (78), thyroideotomized (6), adrenalectomized (6), and castrated (14) White-crowned Sparrows were observed with the electron microscope. Six types of glandular cells were identified and the ultrastructural characteristics of each have been described. To each has been assigned tentatively an endocrine function.STH cells are characterized by the presence of large, dense secretory granules ranging from 220–280 m, a poorly developed endoplasmic reticulum, and a fragmented Golgi apparatus; they occur only in the caudal lobe. They show no remarkable changes after adrenalectomy, castration, and thyroidectomy.Prolactin cells, whose identity is suggested by their responses to photostimulation and surgical experiments, are characterized by large, polymorphic, dense secretory granules; they have been found mainly in the cephalic lobe.ACTH cells, whose function is confirmed by their cytological responses to adrenalectomy, have a peculiar type of secretory granule (220 m) with high and low phases of electron density. They occur exclusively in the cephalic lobe and are transformed, after adrenalectomy to large, vacuolated adrenalectomy cells.TSH cells are so designated by their response to thyroidectomy. After thyroidectomy, they lose their specific fine secretory granules and are transformed into large, vacuolated thyroidectomy cells. We have found TSH cells and thyroidectomy cells only in the cephalic lobe.Two types considered to be gonadotropic cells from their responses to gonadectomy, occur in both the cephalic and caudal lobes. One of them contains spherical, dense secretory granules (180–220 m), prominent rough endoplasmic reticulum and a well developed Golgi apparatus; the other type contains dense secretory granules of variable size (150–350 m), a less extensively developed Golgi apparatus, and sac-like endoplasmic reticulum. Both types of gonadotropic cells show extreme enlargement and vacuolization after castration. However, they retain differences in appearance in the structure of cytoplasmic organelles and vacuolization.Dedicated to Professor Dr. H. Grau in honor of his 70th birthday.The investigation reported herein was supported by a research grant (HE 07240 NEUA) from the National Institutes of Health to Professor Vitums, by a research grant (5R01 NB 06187) from the National Institutes of Health to Professor Farner, and by a scientific research grant (No. 91049) from the Ministry of Education of Japan to Professor Mikami. The authors wish to thank Professor James R. King for his assistance in obtaining and maintaining the birds, and for his helpful advice concerning the experiments.     

Transforming growth factor ß1 acid interaction

Chick embryo skin fibroblasts release transforming growth factor ₁ that is able to modulate glycosaminoglycan synthesis and secretion. When incubated with individual classes of glycosaminoglycans, the factor's modulatory activity was altered. To determine whether direct interactions between transforming growth factor ₁ and glycosaminoglycans occur, we have assessed the activity of the growth factor after pre-incubation with single classes of glycosaminoglycans by assaying its inhibitory effect upon the proliferative response of thymocytes stimulated with interleukin-1. Untreated transforming growth factor ₁ suppressed the proliferative response of thymocytes to interleukin-1, as did transforming growth factor ₁ pre-incubated with sulphated glycosaminoglycans. By contrast, transforming growth factor ₁ lost its inhibitory capacity when preincubated with high molecular weight hyaluronic acid. Digestion of transforming growth factor ₁-hyaluronic acid complex with hyaluronidase released active transforming growth factor ₁. Trypsin degraded transforming growth factor ₁ alone, but did not degrade the transforming growth factor ₁-hyaluronic acid complex. These results suggest that hyaluronic acid interacts with transforming growth factor ₁, thus protecting the factor from tryptic degradation and may be a means of concentrating growth factor activity.     

The effect of monoterpenes on astaxanthin production by a mutant ofPhaffia rhodozyma

Summary The total pigment and astaxanthin content ofPhaffia rhodozyma increased with increasing concentrations -pinene up to 500 l -pinene/l. Above this concentration the total pigment and astaxanthin content as well as the biomass production decreased. The addition of 500 l -pinene/l increased the total pigment content from 1652 g/g to 2201 g/g and the astaxanthin content from 1554 g/g to 1883 g/g. A sharp decrease in maximum specific growth rate occurred above 150 l -pinene/l.     

Micropropagation of flowering dogwood (Cornus florida) from seedlings

A method for regenerating whole plants from nodal (axillary bud) cultures of seedlings was developed for flowering dogwood (Cornus florida L.). The seed source significantly influenced the rate of proliferation, although cultures initiated from each of the seven mother trees produced some shoots. Woody plant medium (WPM) was superior to either Murashige and Skoog or Schenk and Hildebrandt basal medium. 6-Benzyladenine (BA) at 2.2 or 4.4 m stimulated the generation of significantly more useable shoots (1 cm) compared to all other concentrations (0.5–22.5 m tested. Thidiazuron (TDZ) at 0.6 and 1.1 m supported proliferation, but strongly inhibited shoot elongation. TDZ initiated cultures transferred to medium containing 4.4 m BA produced usable shoots after five additional subcultures. Shoots generated adventitious roots when exposed to either a 12-h pulse of relatively high concentrations (246–1230 m or continuous lower concentrations (0.5–49.0 m of indolebutyric acid (IBA) for longer periods. Microshoots produced the significantly greatest number of roots when subjected to 4.9 m IBA in WPM over a 4-week period. Whole plants were acclimatized to the laboratory conditions and subsequently to the greenhouse. The methodology described here should be useful in a breeding program by supplying multiple copies of unique, recombinant genotypes.     

Methylobacterium rhodesianum MB 126 possesses two acetoacetyl-CoA reductases

Two constitutive acetoacetyl-CoA (AcAc-CoA) reductases were purified from Methylobacterium rhodesianum MB 126, an NADPH-linked d(-)--hydroxybutyryl-CoA forming reductase (enzyme A) and an NADH-and NADPH-linked l(+)--hydroxybutyryl-CoA forming reductase (enzyme B). Enzyme A and B give apparent K _m values of 15 M and 30 M for AcAc-CoA, 18 M for NADPH and 30 M for NADH, respectively. They are inhibited by AcAc-CoA at concentrations higher than 25 M and 50 M, respectively. The contribution of the two reductases to poly--hydroxybutyrate synthesis is discussed.     

The importance of PS I chlorophyll red forms in light-harvesting by leaves

We have investigated the importance of the long wavelength absorbing spectral forms (red forms) of Photosystem I in photosynthetic light harvesting by leaves. To this end leaf spectra were simulated by using a linear combination of absorption (OD) spectra of purified Photosystem I, Photosystem II and LHC II, multiplied by an empirical multiple scattering chloroplast/leaf conversion function. In this way it is demonstrated that while the PS I red forms account for only about 4–5% of light absorption in a normal daylight environment, in different shadelight environments these long wavelength pigments may be responsible for up to 40% of total photon capture. In the context of maximising the photosynthetic quantum efficiency under the low light conditions of shadelight, this relative increase in the absorption cross section of PS I can be understood by considering the increased synthesis of the major PS II antenna complex, LHC II, known to occur in plants growing under these light conditions. It is demonstrated that for plants in a moderate to deep shadelight regime the PS II cross section needs to increase by 50% to 100% via LHC II synthesis to balance the increased PS I absorption by the red forms. The possibility that under shade light conditions the increased PS I cross section may serve in cyclic phosphorylation is also discussed.     

Micropropagation of mature Chinese tallow tree (Sapium sebiferum Roxb.)

An in vitro propagation technique based on axillary bud proliferation has been developed for matureSapium sebiferum trees. Nodal segments cultured on Murashige and Skoog (MS) medium supplemented with benzyl adenine (1–10 m and -naphthaleneacetic acid (0–0.5 m showed axillary bud proliferation. Shoots proliferated in vitro were multiplied on Murashige and Skoog medium containing 2.5 m benzyl adenine and 0.25 m -naphthaleneacetic acid. Seasonal changes affected the shoot proliferation potential of the initial explant. Shoots were rooted on a half-strength, growth-regulator-free, agar-gelled, MS medium after a 48-h treatment on half-strength MS liquid medium with 10 m indole-3-butyric acid. Rooted plantlets were potted and acclimatized in a growth chamber and then moved to the greenhouse. Four-month-old plants were transplanted to the field.Abbreviations BA Benzyl adenine - IBA Indole-3-butyric acid - 2-ip N⁶-(-dimethylallylamino)purine - MS Murashige and Skoog (1962) medium - NAA -Naphthaleneacetic acid     

Effects of pyridine nucleotides on the gating of ATP-sensitive potassium channels in insulin-secreting cells

Summary The single-channel current recording technique has been used to study the influences that the pyridine nucleotides NAD, NADH, NADP and NADPH have on the gating of ATP-sensitive K⁺ channels in an insulin-secreting cell line (RINm5F). The effects of the nucleotides were studied at the intracellular surface using either excised inside-out membrane patches or permeabilized cells. All four pyridine nucleotides were found to evoke similar effects. At low concentrations, 100 m and less, each promoted channel opening whereas high concentrations, 500 m and above, evoked channel closure. The degree of K⁺ channel activation by pyridine nucleotides (low conc.) was found to be similar to that evoked by the same concentrations of ADP or GTP, whereas the degree of K⁺ channel inhibition (high conc.) was less marked than that evoked by the same concentrations of ATP, and never resulted in refreshment of K⁺ channels following removal. The effects of NAD, NADH, NADP and NADPH seemed to interact with those of ATP and ADP. In the presence of 1mm ADP and 4mm ATP, 10 to 100 m concentrations of the pyridine nucleotides could not evoke channel opening, whereas concentrations of 500 m and above were found to evoke channel closure. In the presence of 2mm ATP and 0.5mm ADP, however, 10 to 100 m concentrations of the pyridine nucleotides were able to activate K⁺ channels.     

Direct somatic embryogenesis through thin cell layer culture in Panax ginseng

Direct somatic embryos were differentiated on cotyledon transverse Thin Cell Layers (tTCLs) of Panax ginseng after 9 weeks in the Murashige and Skoog basal (MS) medium containing 2,4-d (5M). When MS medium containing 2,4-d (5M) was used for seedling pretreatment and for tTCLs culture, somatic embryos were observed 2 weeks earlier, i.e. after 7 weeks of culture. On the tTCLs from seedlings pretreated with 2,4-d (5M) combined with benzyladenine and zeatin at 0.1 M (BZ), somatic embryos were observed after 6 weeks of culture and the percentage of embryogenesis was higher (62%) than when 2,4-d was used alone for pretreatment (40%). Similar results were also obtained from pretreatment with combinations of 2,4-d (5M) and thidiazuron (TDZ) (0.01, 0.1M). When a combination of 2,4-d (5M) and BZ (0.1M) was used both for seedling pretreatment and for tTCLs culture, both somatic embryos and shoots were observed after only 3 weeks. As the concentration of BZ increased, the percentage of somatic embryogenesis decreased but the percentage of organogenesis increased. Similar responses were obtained with a combination of 2,4-d (5M) and TDZ (0.01M). On the medium containing both NAA (0.3M) and BZ (1M), globular- and heart- stage embryos developed after 4 weeks of culture into cotyledonary-staged embryos which remained dormant after a short elongation of the embryo axis. The importance of seedling pretreatment by growth substances in enhancing somatic embryogenesis is reported.Abbreviations BA 6-benzyladenine - BZ combination of BA and zeatin - 2,4-d 2,4-dichlorophenoxyacetic acid - MS medium Murashige and Skoog basal medium - NAA a-naphthaleneacetic acid - TDZ thidiazuron - tTCLs transverse thin cell layers - TCL longitudinal thin cell layer     

Mitochondrial alpha-glycerol phosphate dehydrogenase activity in IIA fibres of the rat lateral gastrocnemius muscle; the effect of Ca2+ and ATP

Summary Mitochondrial -glycerol phosphate dehydrogenase is an important enzyme, but it is difficult to extract and purify. We have measured the activity of this enzyme in single type IIA skeletal muscle fibres under initial rate conditions by microdensitometry of the formazan reaction product.The K_m (1.6mm) for the substrate (l--glycerol phosphate) was lower than reported for the extracted enzyme. Further, at low substrate concentrations (3mm), the enzyme was allosterically activated by free Ca²⁺ concentrations of 1 m or greater, and half-maximal stimulation occurred at 0.3 m free Ca²⁺. In the absence of Ca²⁺, there was negative cooperativity of substrate binding with a Hill constant of 0.57, but no cooperativity occurred in the presence of calcium. ATP (10mm) inhibited enzyme activity in the presence of Ca²⁺ but not in its absence.