The abnormal phosphorylation of spectrin in human hereditary spherocytosis

The phosphorylation of the proteins of the erythrocyte membrane of patients suffering from hereditary spherocytosis is investigated in intact erythrocytes by their incubation in the presence of radioactive inorganic phosphate. Examination of the phosphorylated components by high-resolution two-dimensional gel electrophoresis reveals only one defect in the pathological membranes, a depressed phosphorylation of the smaller polypeptide of spectrin; band 2. The phosphorylation of band 2 is measured with reference to the phosphorylation of syndein ($\text{2.1 + 2.2 + 2.3}$). In patients showing overt clinical symptoms and for whom splenectomy is advocated the phosphorylation of band 2 is depressed by approx. 70%. After splenectomy the phosphorylation of membrane proteins is restored to normal levels.     

Phototoxicity of phenothiazine derivatives. II. Photosensitized cross-linking of erythrocyte membrane proteins

Irradiation in the presence of O₂, with near-UV light of five promazine (PZ) derivatives added to erythrocyte ghost membranes, causes covalent cross-linking between proteins as revealed by a progressive decrease in the amounts of proteins separable by electrophoresis after denaturation. The induction of cross-links in the two spectrin subunits is a single-hit process as a function of the irradiation time; relatively the rate constants (in min^?1) of the photoreactions were 0.060 with chlorpromazine (CPZ), 0.039 with methoxypromazine (MTPZ), 0.031 with PZ, 0.029 with triflupromazine (TFPZ) and 0.006 with acepromazine (ACPZ).A main photochemical intermediate implicated in the spectrin aggregation seems to be the cation radical of the PZ derivatives. Indeed, (i) the chemically generated cation radicals can induce the reaction in the dark; (ii) the photoaggregation is regularly reduced upon addition of increasing concentrations of NaN₃; (iii) NaN₃ similarly affects the amount of cross-links induced by the isolated cation radicals. Hydroxyl radicals are also involved in the photocross-linking when the reaction is initiated only by MTPZ and not by the other sensitizers.In the absence of oxygen during irradiation, PZ, MTPZ and ACPZ completely loose their cross-linking activities whereas CPZ and TFPZ remain as efficient as in the presence of oxygen.     

Puromycin aminonucleoside increases the synthesis of ribonuclease inhibitor and other proteins in kidney

The incorporation of labelled amino acids into proteins was measured in vivo in the kidney of control rats and rats that received puromycin aminonucleoside. There was an increase in the synthesis of kidney proteins after the aminonucleoside that was similar to the increased synthesis previously observed in cell-free and slice preparations. The increased synthesis in vivo and in vitro especially involved proteins of the prealbumin fraction of average molecular weights of approx. 50 000, 35 000, 25000, 18 000, and 10 000. The largest of these proteins was identified as kidney ribonuclease inhibitor and additional evidence was obtained for the increased synthesis of the kidney inhibitor after aminonucleoside.     

Response of root mRNA population to sulphate shortage in maize seedlings

Roots of ten-days-old seedlings obtained from a maize hybrid grown in complete or in sulphate-deprived medium were used to extract Poly(A)+RNA. The response to sulphate deprivation, which is known to increase the uptake capacity up to ten times, was manifested also by the expression of three mRNA species, as shown by the in vitro translation of the mRNA population. One hour after transfer to complete nutrient medium all three mRNAs were still present.Abbreviations BSA Bovine Serum Albumine - DTT 1,4-Dithio-DL-Threitol - EDTA Ethylenediaminetetraacetic-acid - MES 2-(N-Morpholino) ethane sulfonic acid - SDS Sodium Dodecyl Sulfate - TCA Trichloracetic acid - TRIS Tris(hydroxy-methyl)-aminomethane     

Requirement of trypsin inhibitor for measurement of 3-hydroxy-3-methylglutaryl coenzyme A reductase in intestinal mucosa of rat

A method was developed for the determination of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in the microsomal fraction of crypt cells and villi of rat intestinal mucosa. Addition of trypsin inhibitor to homogenizing and incubation media at a proper concentration appeared inevitable for measurement of the activity of the villi fraction. The reductase in crypt cells was also slightly enhanced by the addition of the inhibitor. Using this technique, the enzyme activity in villi was found to be as active as the crypt cell fraction. Since other types of protease inhibitors were not necessarily effective, it was suggested that specific enzyme(s) inactivates the mucosal reductase in the course of measurement.     

Correction of severe manganese deficiency in wheat with chemical fertilizers

Summary Manganese, N and P fertilizers were applied to wheat in field experiments on a soil so deficient in Mn that it caused the wheat to die before heading. Yields of wheat were increased linearly by soil banded Mn to 44.8 kg/ha, giving a yield of 3.03 tonnes/ha. Yields were increased to a lesser extent by foliar-applied Mn and least by soil-broadcasted Mn. Soil N and P appeared to be adequate, yet ammonium sulphate at 56 kg N/ha where applied alone caused a yield of 1.69 tonnes/ha and ammonium sulphate nitrate gave a yield of 0.98 tonnes/ha, the increases being primarily due to the release of Mn to the plants. Calcium nitrate and triple superphosphate were much less effective in releasing Mn.     

A collagen-like glycoprotein from elastin-rich tissues

Extracts of bovine aorta and nuchal ligament contain several large glycoproteins. The major glycoprotein species has been isolated and has been shown to be collagenase sensitive with an apparent molecular weight of 140,000 daltons. The protein exists in disulphide-bonded aggregates, contains hydroxyproline and hydroxylysine in 1:1 ratio and is unlike any of the known collagen types in amino acid analysis. Its presencein ligament extracts indicates that it is not derived from basement membranes. The evidence suggests that this protein is not derived from the microfibrillar components of the elastic tissues.     

Effect of the natural ATPase inhibitor on the binding of adenine nucleotides and inorganic phosphate to mitochondrial F1-ATPase

(1) Incubation of the beef heart mitochondrial ATPase, F₁ with Mg-ATP was required for the binding of the natural inhibitor, IF₁, to F₁ to form the inactive F₁-IF₁ complex. When F₁ was incubated in the presence of [¹⁴C]ATP and MgCl₂, about 2 mol ¹⁴C-labeled adenine nucleotides were found to bind per mol of F₁; the bound ¹⁴C-labeled nucleotides consisted of [¹⁴C]ADP arising from [¹⁴C]ATP hydrolysis and [¹⁴C]ATP. The ¹⁴C-labeled nucleotide binding was not prevented by IF₁. These data are in agreement with the idea that the formation of the F₁-IF₁ complex requires an appropriate conformation of F₁. (2) The ¹⁴C-labeled adenine nucleotides bound to F₁ following preincubation of F₁ with Mg-[¹⁴C]ATP could be exchanged with added [³H]ADP or [³H]ATP. No exchange occurred between added [³H]ADP or [³H]ATP and the ¹⁴C-labeled adenine nucleotides bound to the F₁-IF₁ complex. These data suggest that the conformation of F₁ in the isolated F₁-IF₁ complex is further modified in such a way that the bound ¹⁴C-labeled nucleotides are no longer available for exchange. (3) ³²P_i was able to bind to isolated F₁ with a stoichiometry of about 1 mol of P_i per mol of F₁ (Penefsky, H.S. (1977) J. Biol. Chem. 252, 2891–2899). There was no binding of ³²P_i to the F₁-IF₁ complex. Thus, not only the nucleotides sites, but also the P_i site, are masked from interaction with external ligands in the isolated F₁-IF₁ complex.