Characterization of DNA kinase from calf thymus

DNA kinase has been purified to homogeneity from calf thymus. The purified enzyme, with a specific activity of 16.7 units/mg protein at 25 degrees C, exhibited a sharp pH/activity curve with a pH optimum at 5.5 and low activity at alkaline pH. The molecular weight of the enzyme was estimated by dodecylsulfate/polyacrylamide gel electrophoresis to be 5.4 X 10(4). The enzyme has a sedimentation coefficient of 4.0 S. An apparent molecular weight of 5.6 X 10(4) and a Stokes' radius of 3.3 nm were estimated by gel-filtration on Sephadex G-100. The enzyme phosphorylates neither yeast RNA nor poly(A) instead of DNA. Compared with rat liver DNA kinase, calf thymus DNA kinase is relatively resistant to the inhibition by sulfate (Ki = 7 mM) and pyrophosphate (Ki = 5 mM). The enzyme activity is markedly stimulated by polyamines at the sub-optimal concentration of Mg2+ but not by monovalent cations.     

Regulatory mechanism of delayed-type hypersensitivity in mice. II. Effect of suppressor cells on the development of memory cells for delayed-type hypersensitivity

Murine lymph node cells (LNC), which we showed previously to noncompetitively inhibit antibody-dependent cellular cytotoxicity (ADCC) to an erythrocyte target, were tested for their ability to inhibit ADCC to a tumor target, EL-4. Both a 4-hr ⁵¹Cr-release cytotoxicity assay and an overnight ¹²⁵IUdR (iododeoxyuridine) postlabeling cytostasis assay were used. Normal autologous lymph node cells inhibited spleen cell-mediated ADCC in both assays. Inhibition by LNC was dose dependent, but comparable numbers of sheep erythrocytes did not inhibit, indicating that LNC-mediated inhibition was not simply a matter of crowding. Inhibitory activity was enriched in LNC after removal of Fc receptor-bearing cells on EA monolayers.     

Purification and subunit structure of rat-liver phosphoprotein phosphatase, whose molecular weight is 260000 by gel filtration (phosphatase IB)

1. Phosphoprotein phosphatase IB is a form of rat liver phosphoprotein phosphatase, distinguished from the previously studied phosphoprotein phosphatase II [Tamura et al. (1980) Eur. J. Biochem. 104, 347-355] by earlier elution from DEAE-cellulose, by higher molecular weight on gel filtration (260000) and by lower activity toward phosphorylase alpha. This enzyme was purified to apparent homogeneity by chromatography on DEAE-cellulose, aminohexyl--Sepharose-4B, histone--Sepharose-4B, protamine--Sepharose-4B and Sephadex G-200. 2. The molecular weight of purified phosphatase IB was 260000 by gel filtration and 185000 from S20,W and Stokes' radius. Using histone phosphatase activity as the reference for comparison, the phosphorylase phosphatase activity of purified phosphatase IB was only one-fifth that of phosphatase II. 3. Sodium dodecyl sulfate gel electrophoresis revealed that phosphatase IB contains three types of subunit, namely alpha, beta and gamma, whose molecular weights are 35000, 69000 and 58000, respectively. The alpha subunit is identical to the alpha subunit of phosphatase II. While the beta subunit is also identical or similar to the beta subunit of phoshatase II, the gamma subunit appears to be unique to phosphatase IB. 4. When purified phosphatase IB was treated with 2-mercaptoethanol at -20 degrees C, the enzyme was dissociated to release the catalytically active alpha subunit. Along with this dissociation, there was a 7.4-fold increase in phosphorylase phosphatase activity; but histone phosphatase activity increased only 1.6-fold. The possible functions of the gamma subunit are discussed in relation to this activation of enzyme.     

Effect of calcium administration on renal responsiveness to parathyroid hormone in pseudohypoparathyroidism type I and II -- in comparison with normals, idiopathic and surgical hypoparathyroidism.

A 31-year-old man and a 12-year-old girl were diagnosed as pseudohypoparathyroidism (PHP) Type I because of a failure to respond to the administration of parathyroid hormone (PTH) with increased urinary excretion of phosphate and cyclic adenosine-3', 5'-monophosphate (cAMP). A 22-year-old woman was diagnosed as PHP Type II because there was no increase in the urinary excretion of phosphate despite of a marked increase in urinary cAMP excretion. With the combined calcium-PTH infusion or PTH infusion after vitamin D therapy, renal response was improved in these patients. Also dibutyryl adenosine-3'-5'-cyclic monophosphate (dbcAMP) infusion evoked an increased urinary phosphate excretion in all of the patients. The metabolic defect of our patients with PHP Type I may be caused not by a lack or defective form of PTH-sensitive receptor adenylate cyclase complex but rather by an abnormal conformation in the plasma membrane-associated receptor adenylate cyclase enzyme complex in kidney. In the patient with PHP Type II, as cAMP generation is intact, the metabolic defect might be related to a defect of calcium mobilization in renal tubular cells in response to PTH.     

A role of membranes in the activation of a new multifunctional protein kinase system.

A new multifunctional protein kinase, which normally exists as an inactive form in the soluble fraction in mammalian tissues, attaches to membranes to exhibit full enzymatic activity. A low concentration of Ca2+ is absolutely necessary for this activation. This process is reversible. cAMP shows no effect. The active factors in membranes are phosphatidylinositol, phosphatidylserine, phosphatidic acid, diphosphatidylglycerol, and phosphatidylethanolamine in that order. Phosphatidylcholine and sphingomyelin are far less effective. Cytoplasmic as well as other membrane fractions from various tissues are active in supporting the enzymatic activity. A possible role of this Ca2+ and phospholipid-activated protein kinase system in transmembrane control is proposed.     

High and selective resistance to mecillinam in adenylate cyclase-deficient or cyclic adenosine 3'',5''-monophosphate receptor protein-deficient mutants of Escherichia coli.

Adenylate cyclase-deficient (cya) mutants of Escherichia coli K-12 were selectively and highly resistant to mecillinam (FL1060) among several beta-lactam antibiotics in the absence of cyclic adenosine 3',5'-monophosphate (cAMP). They became sensitive to the drug in the presence of cAMP. Also, cAMP receptor protein-negative (crp) mutants, with the exception of strain 5333, were highly resistant to mecillinam in the presence and in the absence of cAMP. Mecillinam exerted two distinct and sequential effects in both cya+ strains and cya strains supplemented with cAMP: (i) rounding of cells and (ii) cessation of cell division. The first effect was accompanied by a decrease in growth rate, whereas the second effect was accompanied by enlargement and lysis of the rounded cells. The second effect of mecillinam was dependent on inoculum size and cAMP. When the cell density was above about 10(6) cells per ml, the rounded cells stopped dividing but did not lyse. In the absence of cAMP, cya strains neither stopped dividing nor lysed; they were resistant to the second, lethal effect of mecillinam.     

Optical measurements of intracellular oxygen concentration of rat heart in vitro

The optical characteristics of hemoglobin-free perfused rat heart have been examined in detail. Ethyl hydrogen peroxide is found to convert myoglobin into “ferryl compound” in the perfused heart, as is also seen in vitro. After pretreatment with ethyl hydrogen peroxide, a typical mitochondrial absorption spectrum, similar to that of isolated rat heart mitochondria, is obtained in perfused heart. The overall absorption spectrum of the heart obtained by the aerobic to anaerobic transition is a superposition of the mitochondrial spectrum on that of myoglobin. By comparing these spectra, it is found that measurement of cytochrome a + a₃ at 605–620 nm is possible in spite of the absorbance change due to the oxygenation-deoxygenation of myoglobin, whereas the wavelength pairs for cytochrome c at 550-540 nm, cytochrome b at 562–575 nm and cytochrome a + a₃ at 445–450 nm can not be used in the heart because of interference from the absorption change of myoglobin. The partial pressure of O₂ (P₅₀) which is required for half maximal deoxygenation (or oxygenation) of myoglobin in perfused heart is found to be 2.4 mm Hg at room temperature and the Hill constant, n, is 1.1; these values are similar to those of myoglobin purified from rat heart. The steady-state O₂ titration has been performed by using absorbancy changes of myoglobin and cytochrome a + a₃ as intracellular O₂ indicators. In the perfused heart, the percentage change of oxygenation-deoxygenation of myoglobin parallels the oxidation-reduction of cytochrome a + a₃, while the mixture of purified myoglobin and isolated mitochondria shows a deviation, reflecting the difference of O₂ affinities between myoglobin and cytochrome a + a₃. The results indicate that there may be an O₂ gradient between cytosolic and mitochondrial compartments in the hemoglobin-free perfused heart. The absorption changes of myoglobin and of cytochrome a + a₃ can be measured in a single contraction-relaxation cycle. A triple beam method was introduced to eliminate the effect of light scattering changes in these measurements. The results demonstrated that myoglobin is more oxygenated during the systolic and diastolic periods and deoxygenated in the resting period, whereas cytochrome a + a₃ is more reduced in systole and diastole and oxidized in the resting state. Changing the perfusion conditions greatly alters the time course of the events which occur during the contraction-relaxation cycle of the perfused heart.     

Tunicamycin-resistant mutants and chromosomal locations of mutational sites in Bacillus subtilis.

The types of tunicamycin-resistant mutants of Bacillus subtilis were analyzed, and their mutational sites on the chromosome were mapped. A type 1 mutation that simultaneously expressed hyperproductivity of extracellular alpha-amylase was located close to amy E. Type 2 mutations were near aroI.     

Fatty acid desaturase system of yeast microsomes. Involvement of cytochrome b5-containing electron-transport chain.

The oxidative desaturation of palmitoyl CoA by microsomes from anaerobically grown Saccharomyces cerevisiae has been studied by using NADH as electron donor. The desaturation product was identified as palmitoleic acid by periodate oxidation. The desaturase activity was sensitive to relatively high concentrations of cyanide; the concentration of cyanide causing half-maximal inhibition was determined to be 7.1 mm. The rate of reoxidation of cytochrome b₅ in NADH-reduced microsomes was stimulated by the addition of palmitoyl CoA, and the amount of cytochrome b₅ reoxidized by the palmitoyl CoA added could be closely correlated to the amount of palmitoleate formed. No stimulation of the reoxidation of cytochrome b₅ was induced by palmitoyl CoA in microsomes prepared from the desaturase-repressed cells and from a desaturase-deficient mutant, strain KD-20. It is concluded that the fatty acyl CoA desaturase system of yeast microsomes involves cytochrome b₅ as an electron carrier and that the terminal desaturase is sensitive to relatively high concentrations of cyanide.     

The mechanism of the movement of leucocytes

In a study of the movement of human leucocytes it was clarified that characteristic contraction waves were observed on the cell surface during movement and an initial morphological change directly related to the appearance of the wave originated in the surface of the granuloplasm and not in the cell membrane. From these findings, together with physicochemical properties of the contractile protein from equine leucocytes, it was proposed that the wave observed in moving leucocytes might be conducted, in some way, by contraction and relaxation of the contractile protein in the cells. Myosin A and actin as constituents of the contractile protein were extracted separately from leucocytes in polymerized form, which resemble myosin aggregate and F-actin from muscle, respectively. The thick and thin filaments of about 150 and 80 Å in diameter were observed in glycerinated leucocytes with electron microscopy. When glycerinated leucocytes were incubated with heavy meromyosin (HMM) from rabbit skeletal myosin A, the thin filaments developed a structure resembling the ‘arrowhead structure’ of the HMM F-actin complex in vitro. The thick filaments seemed to correspond to myosin aggregates and the thin ones to filaments containing F-actin.