Isolation and characterization of an abundant and novel 22-kDa protein (SM22) from chicken gizzard smooth muscle

Chicken gizzard smooth muscle contains a highly abundant protein (SM22) with an apparent Mr on sodium dodecyl sulfate-polyacrylamide electrophoretic gels of 23,000. The ratio of actin:SM22:tropomyosin in this tissue is estimated to be 6.5(+/- 0.8):2.0(+/- 0.2):1.0. At least three isoelectric isoforms are present in ratios of alpha:beta:gamma of 14:5:1 with alpha the most basic and gamma the most acidic. A method for the purification of SM22 and partial separation of its isoforms is described. Amino acid analyses of purified alpha and beta demonstrate the presence of 1 and 2 half-cystines, respectively, and a lower content of basic amino acids in beta. A value of 22,000 for the Mr of alpha estimated by sedimentation equilibrium indicated its presence as a monomer at physiological ionic strengths. Estimates of the translational frictional coefficient (f/fmin) of alpha calculated from its Stokes radius (25.5 A) and Mr were consistent with its existence as a moderately asymmetric globular protein. Calculations based on its far-ultraviolet CD spectrum provided values of 37% alpha-helix, 31% beta-sheet, 5% beta-turn, and 27% random coil. SM22 was shown not to share functional properties with several proteins of similar Mr and isoelectric point such as myokinase, brain 23-kDa protein, and troponin I. We conclude that it is a novel protein not previously isolated or characterized from any tissue.     

The role of blood platelets in nucleoside metabolism: assay, cellular location and significance of thymidine phosphorylase in human blood

The enzyme thymidine phosphorylase (thymidine: orthophosphate deoxyribosyltransferase, EC 2.4.2.4.), which plays a crucial role in nucleic acid metabolism in both prokaryotic and eukaryotic cells by regulating the availability of thymidine, is present in mammalian blood. Here we describe a simple, rapid HPLC-based micromethod for the assay of blood thymidine phosphorylase. We have arbitarily defined 1 unit of blood thymidine phosphorylase activity as the activity required to produce a 1-nM increment in the plasma concentration of thymine after incubation for 1 h at 37°C with a saturating concentration of exogenous thymidine.

In normal adults, whole (peripheral venous) blood thymidine phosphorylase activity with blood cells intact was 64 ± 11 units (mean ± S.D., n =20, range 45–89). The apparent Michaelis constant for thymidine was of the order to 10⁻⁴ M but varied nearly 5-fold between different individuals. Activity increased when blood cells were permeabilised or lysed with non-ionic detergents, implying that thymidine phosphorylase is an intracellular enzyme which may be influenced by exogenous as well as intracellular factors. When blood from normal donors was fractionated, thymidine phosphorylase activity consistently co-isolated with platelets. Whole-blood thymidine phosphorylase activity correlated well with platelet parameters. Although thymidine phosphorylase activity was also detected in plasma and serum, the small size and notorious fragility of platelets suggest its platelet origin.

Blood from leukaemic donors showed significantly increased thymidine phosphorylase activity compared to normal controls (mean activity ± S.D. was 96 ± 27 units; range 58–140, n = 8).

Thymine formation from thymidine was temperature- and pH-depdendent in whole blood. 2′-Deoxyuridine and 3 of its 5-halogenated analogues (but not 3′-azido-3′-deoxythymidine (AZT), were catabolised by blood thymidine phosphorylase, even during blood clotting at room temperature. Assumptions about in vivo concentrations of these compounds should therefore be interpreted cautiously.

In the presence of high concentrations of thymine and suitable deoxyribose donors, small amounts of thymidine were formed in some blood samples, so it is conceivable that thymidine catabolism may be reversible in vivo under some circumstances.     

Influence of Drought Acclimation and CO(2) Enrichment on Osmotic Adjustment and Chlorophyll a Fluorescence of Sunflower during Drought

Osmotic adjustment occurred during drought in expanded leaves of sunflowers (Helianthus annuus var Hysun 30) which had been continuously exposed to 660 microliters CO₂ per liter or had been previously acclimated to drought. The effect was greatest when the treatments were combined and was negligible in nonacclimated plants grown at 340 microliters CO₂ per liter. The concentrations of ethanol soluble sugars and potassium increased during drought but they did not account for the osmotic adjustment. The delay in the decline in conductance and relative water content and in the loss of structural integrity with increasing drought was dependent on the degree of osmotic adjustment. Where it was greatest, conductance fell from 5.8 millimeters per second on the first day of drought to 1.3 millimeters per second on the fourth day and was at approximately the same level on the eighth day. The relative water content remained constant at 85% for three days and fell to 36% on the sixth day. There was no evidence of leaf desiccation even on the eighth day. In contrast, the conductance of leaves showing minimal adjustment fell rapidly after the first day of drought and was negligible after the fourth, at which time the relative water content was 36%. By the sixth day of drought, areas near the margins of the leaves were desiccating and the plants did not recover upon rewatering. Despite the differences in the rate of change of conductance and relative water content during drought, photosynthetic electron transport activity, inferred from measurements of chlorophyll a fluorescence in vivo and PSII activity of isolated thylakoids, remained functional until desiccation occurred.     

Excimer fluorescence of equine platelet tropomyosin labeled with N-(1-pyrenyl)iodoacetamide

Tropomyosin from equine platelets was reacted with N-(1-pyrenyl)iodoacetamide, a sulfhydryl-specific fluorescent reagent, to give an average extent of incorporation of 1.12 pyrene (Py) groups per platelet tropomyosin (P-TM) chain. The predominant site of reaction on P-TM was the penultimate COOH-terminal residue, Cys-246. The high proportion of the total emission that is due to pyrene ecximers and the pretransition observed in thermal denaturation of Py-P-TM point to a rather loose structure for the COOH-terminal amino acid residues of P-TM. The label on Cys-246 also reports on end-to-end overlap interactions that occur between two different tropomyosin molecules. Additions to a Py-P-TM solution at low ionic strength of unlabeled P-TM, rabbit cardiac tropomyosin (C-TM), or a carboxypeptidase A treated, nonpolymerizable derivative of C-TM all reduce the extent of excimer fluorescence from the sample. Addition of salt greatly reduces the effects of the unlabeled TM species on the Py-P-TM emission spectrum. Circular dichroism measurements indicate Py-P-TM still to be greater than 95% helical. However, analysis of excimer fluorescence levels in samples that contained a constant protein concentration but different mole ratios of labeled to unlabeled P-TM suggests that the bulky pyrene group may diminish the tendency of Py-P-TM to polymerize in an end-to-end manner.     

An HLA-C-specific DNA probe

Analysis of available nucleotide sequence data for class I HLA genes has established that the seventh intron is one of the gene regions which expresses the highest degree of locus specificity (the percentage sequence divergence between nonallelic genes minus the percentage sequence divergence between allelic genes). We have subcloned short DNA sequences including this region from the HLA-Cw3 gene. Two clones, pC250 and pC800, were tested by hybridizing them at high stringency to a panel of clones containing class I HLA genes. Under conditions permitting a strong hybridization signal with a C-locus gene, pC800 also expressed a weak but significant hybridization to other class I genes, while pC250 appeared to hybridize exclusively to the C-locus gene. Hybridization of the pC250 probe at high stringency to Hind III-digested genomic DNA from a panel of unrelated individuals and homozygous typing cell lines revealed a single band in all cases. However, equivalent hybridization against Eco RI-digested DNA revealed two hybridization bands, one at 7.9 kb which correlated with the serologically defined Cw5 and Cw8 alleles, and one at 7.6 kb which correlated with the Cw1, Cw2, Cw3, Cw4, Cw6, and Cw7 alleles.     

Stress tolerance and stress-induced injury in crop plants measured by chlorophyll fluorescence in vivo: chilling, freezing, ice cover, heat, and high light

The proposition is examined that measurements of chlorophyll fluorescence in vivo can be used to monitor cellular injury caused by environmental stresses rapidly and nondestructively and to determine the relative stress tolerances of different species. Stress responses of leaf tissue were measured by F_R, the maximal rate of the induced rise in chlorophyll fluorescence. The time taken for F_R to decrease by 50% in leaves at 0°C was used as a measure of chilling tolerance. This value was 4.3 hours for chilling-sensitive cucumber. In contrast, F_R decreased very slowly in cucumber leaves at 10°C or in chilling-tolerant cabbage leaves at 0°C. Long-term changes in F_R of barley, wheat, and rye leaves kept at 0°C were different in frost-hardened and unhardened material and in the latter appeared to be correlated to plant frost tolerance. To simulate damage caused by a thick ice cover, wheat leaves were placed at 0°C under N₂. Kharkov wheat, a variety tolerant of ice encapsulation, showed a slower decrease in F_R than Gatcher, a spring wheat. Relative heat tolerance was also indicated by the decrease in F_R in heated leaves while changes in vivo resulting from photoinhibition, ultraviolet radiation, and photobleaching can also be measured.     

“Unblocking” Serum Activity <Emphasis Type="Italic">in vitro</Emphasis> in the Polyoma System may Correlate with Antitumour Effects of Antiserum <Emphasis Type="Italic">in vivo</Emphasis>

In a variety of tumour systems, individuals carrying progressively growing neoplasms have lymphoid cells with a specific cytotoxic effect on cultured tumour cells from the same individual^1–4. Since the sera of tumour-bearing individuals have been shown to prevent tumour cell destruction by immune lymphocytes in vitro^2,5–8 and since this serum blocking activity appears early in primary and transplant tumour development^5,7, it has been suggested that the appearance of this serum blocking activity might be responsible for the progressive growth of tumours in individuals having cytotoxic lymphocytes. Counteraction of this blocking activity would thus be of primary importance in facilitating the function of an already existing or bolstered cell-mediated immunity. The serum blocking activity might be inhibited in various ways, by preventing the formation of blocking antibody or by interfering with its action (“unblocking”), as demonstrated in Moloney sarcoma regressor sera⁹. This type of serum also has a therapeutic effect on Moloney sarcomas in vivo^10,11, which has been tentatively attributed to its unblocking activity^8,9 or, possibly, to a complement-dependent cytotoxicity¹⁰. Tumour growth in the Moloney sarcoma system, however, might be due in part to continuous recruitment of neoplastic cells by virus-induced transformation and so the therapeutic effect could be due to a virus-neutralizing serum activity^9,10.     

On the mechanism of ATP-induced shape changes in the human erythrocyte membranes: the role of ATP

In the preceding paper (Sheetz, M. and S.J. Singer. 1977. J Cell Biol. 73:638-646) it was shown that erythrocyte ghosts undergo pronounced shape changes in the presence of mg-ATP. The biochemical effects of the action of ATP are herein examined. The biochemical effects of the action of ATP are herein examined. Phosphorylation by ATP of spectrin component 2 of the erythrocyte membrane is known to occur. We have shown that it is only membrane protein that is significantly phosphorylated under the conditions where the shape changes are produced. The extent of this phosphorylation rises with increasing ATP concentration, reaching nearly 1 mol phosphoryle group per mole of component 2 at 8mM ATP. Most of this phosphorylation appears to occur at a single site on the protein molecule, according to cyanogen bromide peptide cleavage experiments. The degree of phosphorylation of component 2 is apparently also regulated by a membrane-bound protein phosphatase. This activity can be demonstrated in erythrocyte ghosts prepared from intact cells prelabeled with [(32)P]phosphate. In addition to the phosphorylation of component 2, some phosphorylation of lipids, mainly of phosphatidylinositol, is also known to occur. The ghost shape changes are, however, shown to be correlated with the degree of phosphorylation of component 2. In such experiment, the incorporation of exogenous phosphatases into ghosts reversed the shape changes produced by ATP, or by the membrane-intercalating drug chlorpromazine. The results obtained in this and the preceding paper are consistent with the proposal that the erythrocyte membrane possesses kinase and phosphates activities which produce phosphorylation and dephosphorylation of a specific site on spectrin component 2 molecules; the steady-state level of this phosphorylation regulates the structural state of the spectrin complex on the cytoplasmic surface of the membrane, which in turn exerts an important control on the shape of the cell.