Genetic variation in the carbonic anhydrase isozymes of macaque monkeys. IV. Degradation by heat and proteolysis of normal and variant carbonic anhydrase isozymes of Macaca nemestrina

Studies were undertaken on the heat denaturation and proteolytic degradation by alpha-chymotrypsin of the normal red cell carbonic anhydrase isozyme, CA II, and two electrophoretic variants of carbonic anhydrase I, CA Ia and CA Ib, of the pigtail macaque. The heat degradation results showed a difference of about 40-fold in the rate constants between CA Ia and CA Ib, which is due to the marked thermostability of CA Ib compared to CA Ia. The enthalpies and entropies of activation were calculated from the heat denaturation constants. These values were compared, on enthalpy-entropy compensation plots, with those values previously determined for the human CA I and CA II isozymes. They were highly correlated and clearly fell into two distinct clusters, separated by about 200 kJ mol-1; one group comprising the macaque and human CA I isozymes and the other the CA II isozymes. The proteolytic degradation results showed that CA Ia is degraded about 2.5 times more rapidly than CA Ib by alpha-chymotrypsin. Thus, the characteristic 3/1 ratio of CA Ib/CA Ia in mature red cells could be accounted for by the greater susceptibility of CA Ia to degradation at some stage in red cell development.     

The amino acid sequence of carbonic anhydrase I from the rhesus macaque

The complete amino acid sequence of carbonic anhydrase I (CA I) isolated from the red cells of the rhesus macaque ($\text{Macaca}$$\text{mulatta}$) is presented. This sequence was obtained by aligning peptides derived from various fragmentation procedures with the fully characterized sequence of human CA I. When the peptides of rhesus CA I were ordered in this manner, 13 of the 260 residues were found to differ from the human CA I sequence. The known markedly higher specific esterase activity of rhesus CA I compared to human CA I could not be correlated with any changes in residues postulated to be within 10 Å of the single zinc ion at the active site.     

24,24-Difluoro-25-hydroxyvitamin D3-enhanced bone mineralization in rats. Comparison with 25-hydroxyvitamin3 and vitamin D3

The biological activity of 24,24-difluoro-25-hydroxyvitamin D₃ was assessed using elevation of serum phosphorus and healing of rickets of vitamin D-deficient rats. Various levels of 24,24-difluoro-25-hydroxyvitamin D₃ and 25-hydroxyvitamin D₃ were administered daily for 2 weeks in the dose range of 6.5 to 3250 pmol after feeding rats a low phosphorus, vitamin D-deficient diet for 3 weeks. Vitamin D₃ was concurrently tested at dose levels of 650 and 3250 pmol. 24,24-Difluoro-25-hydroxyvitamin D₃ is approximately equipotent with 25-hydroxyvitamin D₃ in stimulation of growth, mineralization of rachitic bone, and elevation of serum inorganic phosphorus. Radiological manifestations of rickets were also equally improved by 24,24-difluoro-25-hydroxyvitamin D₃ and 25-hydroxyvitamin D₃. Compared with vitamin D₃, these compounds were approximately 5 to 10 times more active in mineralization using rats on a low phosphorus, vitamin D-deficient diet. The functional role, if any, for 24-hydroxylated vitamin D compounds, such as 24,25-dihydroxyvitamin D₃, therefore remains obscure. It appears that vitamin D compounds that cannot be 24-hydroxylated evoke no disorder in bone mineralization.     

Regulation of aspartate aminotransferase activity associated with change of pyridoxal phosphate level.

The activities of aspartate aminotransferase (EC 2.6.1.1) in the cytosol fractions of the liver and kidney of rats fed pyridoxine-deficient or control diet for 3 weeks were determined. In the absence of pyridoxal phosphate, the activities in the liver and kidney preparations of deficient rats were both abnormally low. The activity in the kidney fraction of deficient rats was restored to almost the control level by addition of pyridoxal phosphate, whereas that of the liver was only partially restored. The antigen activity, however, measured using anti-aspartate aminotransferase, was similar in liver fractions from deficient and control rats. These findings suggest the existence of a form of transaminase with little or no activity in the liver of deficient rats. The properties of the crude enzymes from deficient and control rats were indistinguishable by immunodiffusion, and the enzymes had the same subunit size and heat stability under the conditions tested. However, purified enzyme from deficient rat liver had a different specific activity and absorption spectrum from purified enzyme from normal liver.     

Distribution of IP25 in chromatin and its possible involvement in chromatin condensation

Friend leukaemic cells (FLC) were induced to differentiate with dimethyl sulfoxyde (DMSO), hexamethylenbis-acetamide (HMBA) and sodium butyrate (SB) and the phospholipid composition was analyzed. The phospholipid composition of differentiated cells differed from that of non differentiated cells and also varied according to inducer. The ratios of the percentage of phosphatidyl choline (PC) to that of phosphatidyl ethanolamine (PE) or sphingomyelin (SPH) increased by about 2-fold in DMSO or SB induced FLC. These ratios did not vary in HMBa induced FLC. Furthermore the fatty acid composition of PC and PE obtained from differentiated cells varied according to the inducer. Although these changes appeared to be related to the inducers, it can not be excluded that the differentiated state also contributes to these changes.     

Formation of a carboxyarabinitol bisphosphate complex with ribulose bisphosphate carboxylase/oxygenase and theoretical specific activity of the enzyme

¹⁴C-Labeled 2-carboxyarabinitol-1,5-bisphosphate was bound to both nonactivated and CO₂and Mg²⁺ activated forms of ribulose bisphosphate carboxylase/oxygenase. The complex could be precipitated with 20% polyethylene glycol and 20 mm MgCl₂ for quantitation of the moles of the affinity label bound per mole of enzyme. The [¹⁴C]carboxyarabinitol-P₂ bound to the nonactivated enzyme could be exchanged with a 100-fold excess of the unlabeled compound. With the activated enzyme the binding of [¹⁴C]carboxyarabinitol-P₂ was so tight that it did not exchange with the unlabeled compound and a binding stoichiometry of one molecule per active site was assumed. This tight binding was dependent upon pretreatment of the enzyme with both CO₂ and MgCl₂ in the same manner that enzyme activation depended on CO₂ and Mg²⁺ concentrations. Various enzyme preparations from spinach leaves tightly bound [¹⁴C]carboxyarabinitol-P₂ in proportion to their specific activities. By extrapolating to a maximum binding of 8 mol of [¹⁴C]carboxyarabinitol-P₂ per mole of this A₈B₈ enzyme a theoretical specific activity of 2.8 μmol · min^?1 · mg protein^?1 was indicated. Enzyme preparations purified from spinach leaves generally have a specific activity in the range of 1.0 to 2.3.     

Biosynthesis of alpha-galactosidase A in cultured Chang liver cells

An investigation of the structure and biosynthesis of alpha-galactosidase A (alpha-D-galactoside glycohydrolase, EC 3.2.1.22) and its N-linked oligosaccharide chains was undertaken by metabolic labeling of Chang liver cells with [2-3H]mannose, immunoprecipitation of the activity, and examination of the resulting immunoprecipitates. From cells pulse labeled for 3 h, two radioactive bands with Mr = 58,000 and 49,000 were detected by SDS-gel electrophoresis; following a 20-h chase, only the Mr = 49,000 band was observed. Examination of the oligosaccharide fraction derived from pulse-labeled enzyme revealed that 18% of the asparagine-linked oligosaccharides were complex and 82% were high-mannose type. After a 20-h chase, 48% of the oligosaccharides were complex and 52% were high mannose. The high-mannose oligosaccharides of alpha-galactosidase A immunoprecipitated from both pulsed and pulse-chased cells had the same mobilities as Man8-9GlcNAc on thin-layer chromatography and Bio-Gel P-4. Two fractions of complex glycopeptides derived from the alpha-galactosidase A of pulsed and pulse-chased cells had the same migration on Bio-Gel P-4 as glucose oligomers containing 14 and 19-39 glucose units. Based on their apparent size and their behavior on concanavalin A-Sepharose, the complex oligosaccharides are believed to be composed of tri- and/or tetraantennary structures.     

Reducible cross-links in human glomerular basement membrane

Reducible cross-links in purified human glomerular basement membrane (GBM) were examined with an ion exchange chromatographic system that provided complete separation of cross-link standards and glucosylamines. After hydration in phosphate buffer, lyophilized GBM was reduced with tritiated borohydride. Chromatographic separation revealed two major radioactive peaks, identified as di-hydroxylysinonorleucine (di-OHLNL) and hydroxyaldolhistidine (HAH) by coelution with authentic di-OHLNL and HAH standards. Radioactive glucitol-lysine and glucitol-hydroxylysine were also identified on the basis of their co-elution with synthetic standards. The findings document the existence and establish the nature of the major reducible cross-links in adult human GBM.     

Further studies of the selective inhibition by ouabain of antigen-induced proliferation of human lymphocytes

Cultures of human lymphocytes incubated for 48 hr in the presence of 2 × 10^?7M solutions of the cardiotonic steroid ouabain lose the proliferative response to antigens (SL-0, SK-SD) but can still proliferate when stimulated by nonspecific mitogens (PHA, Con A, pokeweed mitogen). The two-way mixed lymphocyte reaction was also irreversibly lost if cells of both donors were subjected to ouabain pretreatment. Neither cell counts nor cell viability (determined by dye exclusion) were significantly affected by the ouabain treatment. Pretreatment of a suspension of macrophages with the cardiac glycoside did not diminish their capacity to restore the proliferative response to antigen of macrophage-depleted lymphocyte suspensions; on the other hand, untreated macrophages could not restore the proliferative response of cultures of ouabain-pretreated lymphocytes. The ouabain treatment did not change the proportion of cells able to bind fluorescent anti-immunoglobulin nor did it modify the proportion of lymphocytes forming rosettes with either untreated, or antibody coated, red cells. Increased concentration of K⁺ in the medium, either during or after the ouabain treatment, did not reduce the ouabain effect. We conclude that the selective loss of certain lymphocyte functions caused by ouabain pretreatment was due to an effect on the lymphocyte and not on the macrophage; the effect was not due to the elimination of a relatively large fraction of the cells nor to a generalized disappearance of membrane antigens and receptors.     

The sequence of an atriopeptigen: A precursor of the bioactive atrial peptides

The high molecular weight fraction (atriopeptigen-APG) obtained by gel filtration chromatography of rat atrial extracts was fractionated by isoelectric focusing and reverse phase HPLC to obtain a pure APG. Purification of cyanogen bromide digests of the crude high molecular weight fraction resulted in the isolation of a single biologically active cyanogen bromide cleavage peptide. Sequence analyses of these peptides coupled with recent reports of sequence analyses of intermediate molecular weight atrial peptides (Thibault, et al. (1984) FEBS Letters 167, 352–356, and Kangwa, et al., Biochem. Biophys. Res. Commun 119, 933–940) provide the complete primary structure of an 111 residue APG.     

Regulation of plasma membrane Ca2+ ATPases by lipids of the phosphatidylinositol cycle

When the erythrocyte plasma membrane Ca2+ pump is reconstituted into phosphatidylcholine liposomes, the inclusion of small amounts of phosphatidic acid or phosphatidylinositol 4,5-bisphosphate stimulates the enzyme's activity. Other lipids of the phosphatidylinositol cycle (diacylglycerol, phosphatidylinositol) have little effect. The stimulatory effect of phosphatidylinositol 4,5-bisphosphate is greater than that of calmodulin; this lipid also stimulates the plasma membrane Ca2+ ATPase from rat brain.     

Biosynthesis of phytoene from isopentenyl pyrophosphate by a Neurospora enzyme system.

An enzyme system catalyzing the conversion of isopentenyl pyrophosphate to phytoene has been isolated from Neurospora crassa mycelia. This enzyme system shows an absolute requirement for Mg^?, but no other cofactors. Cultures of N. crassa exhibit a low level of phytoene synthesizing activity when grown in the dark. A 2-min in vivo blue light irradiation results in a ninefold increase in activity after 24 h. This increase is dependent on the duration of the light treatment and is inhibited by cycloheximide. A similar blue light-induced elevation of phytoene synthesizing activity was demonstrated in an albino-1 mutant. This enzyme activity was not found in either dark-grown or irradiated cultures of an albino-2 or an albino-3 mutant.     

Regulation and intracellular localization of the biotin holocarboxylase synthetase of 3T3-L1 cells

A quantitative assay has been developed to measure holocarboxylase synthetase activity in cellular extracts. This assay was based on measuring the incorporation of [3H]biotin of high specific activity (4.3 Ci/mmol) into purified rat liver apopyruvate carboxylase. With this assay, holocarboxylase synthetase in 3T3-L1 mouse fibroblasts has been monitored. During the differentiation of this cell from a fibroblast to an adipocyte, holocarboxylase synthetase activity was found to increase threefold, while pyruvate carboxylase activity rose 20-fold. The results suggest a possible relationship between the activity of the holocarboxylase synthetase and the level of the biotin-dependent carboxylases within the mammalian cell. Utilizing digitonin fractionation. the intracellular distribution of this enzyme has also been examined. In the 3T3-L1 cell, the large majority (approximately 70%) of the total holocarboxylase synthetase activity was found in the cytosolic compartment.     

Comparative analysis of the heterogeneity of mononuclear cells present in adult and cord blood by simultaneous examination of multiple phenotypic characteristics

In an attempt to better relate specific membrane characteristics of human adult and cord blood lymphocytes to specific functional activities, the phenotypic differences that exist in these two populations have been examined. Cord blood cells have considerably more spontaneous suppressor cell activity than adult cells. A technique that allows cells to be examined simultaneously for their ability to ingest latex beads, react with specific monoclonal antisera, bind sheep erythrocytes, or react with the Fc portion of IgG was used. As well as assessing fresh populations, phenotypic changes that occur when such cells are held in culture or stimulated with phytohemagglutinin for 3 days were sought. Many differences were found when comparing these mononuclear populations. These included the observations that 12% of adult and 9% of cord blood E-rosette-forming cells ingest latex beads and that 9% of OKT3 reactive cells in both populations did not form E rosettes. In cord blood 58% of T cells that bind OKT8 do not form E rosettes. A similar percentage of cord blood T8-positive cells express a receptor for Fc gamma, such cells being very uncommon in adult blood. Four "monocyte" subpopulations were identified in both samples. One such population (an OKM1- and Fc gamma-positive, nonphagocytic cell) was three times more common in cord blood. In cord blood some OKM1-positive cells also appeared to be simultaneously OKT8 positive. These phenotypic variations forward populations that may be candidates responsible for the functional differences noted in vitro.     

Effects of hypophysectomy, adrenalectomy and corticosterone treatment on uptake and release of putative central neurotransmitters by rat hypothalamic tissue in vitro.

Uptake and release of radiolabeled serotonin, noradrenaline, dopamine, acetylcholine and GABA by rat hypothalamic tissue $\text{in virto}$ were examined following various treatments, which cause drastic changes in the tissue levels of corticosterone. Hypophysectomy affected both uptake and release of most of the neurotransmitters studied. However, adrenalectomy had a more selective effect, changing these processes for serotonin only. The uptake of radiolabeled serotonin by synaptosomes was decreased by about 30%, while its release from tissue slices upon depolarization with 40 mM K⁺ was increased 25%. Both of these changes could be prevented by injecting adrenalectomized rats with corticosterone.It is suggested that corticosteroid hormones might play a modulatory role in maintaining a certain functional activity level in central serotonergic neurons.     

H-2 restriction specificity of T cells from H-2 incompatible radiation bone marrow chimeras: Further evidence for the absence of crucial influence of the host/thymus environment on the generation of H-2 restricted TNP-specific T lymphocyte precursors

Experiments were conducted to answer the questions related to (a) the role played by the antigen-presenting cells (APCs) present within the thymus and (b) the effect of radiation dose to the recipients on the H-2 restriction profile of TNP-specific cytotoxic T lymphocyte precursors (CTLP) recovered from spleens and/or thymuses of H-2 incompatible radiation bone marrow chimeras (BMC). The H-2 restriction profile of intrathymically differentiating TNP-specific CTLPs was also analyzed in order to test an argument that donor-H-2 restricted CTLP detected in spleens of H-2 incompatible BMC were due to the extrathymically differentiated T cells under the influence of donor-derived lymphoreticular cells. The results indicated the following: (i) splenic T cells from B10(H-2^b)→ (B10(H-2^b) → B10.BR(H-2^k)) chimeras, which were constructed by irradiating primary BIO → B10.BR chimeras with 1100 R and reconstituting them with donor-type (B10) bone marrow cells as long as 8 months after their construction, manifested restriction specificities for both donor- and host-type H-2, (ii) splenic T cells from two types of (B10 × B10.BR)F₁→ B10 chimeras which were reconstituted after exposure of the recipients with either 900 or 1100 R with donor-type bone marrow cells generated both donor- and host-H-2 restricted TNP-specific cytotoxic T cells, and (iii) the TNP-specific CTLPs present in the regenerating thymuses of B10.BR → B10 and (B10 × B10.BR)F₁→ B10 chimeras 4 weeks after their construction were also shown to manifest both donor- and host-H-2 restriction specificities. The significance of these findings on the H-2 restriction profile of CTLP generated in BMCs is discussed.     

A bridged Mo(VI) complex containing six and five coordinate Mo: Synthesis, 95Mo N.m.r. spectroscopy and X-ray crystal structure of Mo2O5(C17H17N2O)2

The kinetics of the reaction between Carcinus maenas hemocyanin and cyanide has been studied at various KCN concentrations and a different temperatures (21° and 4°C) by following the decrease of the copper-peroxide absorption band, centered at 337 nm, of the copper still bound to the protein and the intrinsic fluorescence changes as functions of time. In all conditions used, the absorption band completely disappears and KCN concentration affects only the rate of the process. The reaction is kinetically homogeneous indicating no site-site interaction. The apparent rate constant increases with the square of cyanide concentration and the inverse of O₂ concentration. The copper still bound decreases at a rate slower than the 337 nm absorption and the process is not kinetically homogeneous. The fluorescence of the protein increases after an induction period showing an inflection point at about 50% of the total effect. A kinetic model has been proposed on the assumption that the two metal ions are removed sequentially from the active site. The experimental data are in agreement with the theoretical equations derived from the model. The equilibrium constants for the formation of the complex between the first and the second copper ion with cyanide and the rate constants of their decomposition have been calculated. The rate-limiting process for the removal of the second copper ion is the formation of the complex with cyanide.     

Terminal deoxynucleotidyl transferase activity in a cell line (molt-4) derived from the peripheral blood of a patient with acute lymphoblastic leukemia

A terminal deoxynucleotidyl transferase having a sedimentation coefficient of 3–4S has been found associated with the chromatin from a cell line (Molt-4) derived from the peripheral blood of a patient with acute lymphoblastic leukemia.     

Transfer of memory cells into antigen-pretreated hosts. I. Functional detection of migration sites for antigen-specific B cells.

The distribution of functionally active hapten-specific B memory cells was investigated. Using antigen-pretreated lethally irradiated recipients, a marked accumulation of adoptively transferred B memory cells was demonstrated in lymph nodes containing specific antigen, but not in lymph nodes containing non-cross-reacting hapten conjugates. This difference in responsiveness between lymph nodes containing specific versus those containing nonspecific antigen developed over a period 3–5 days after memory cell transfer. The localization of antigen specific cells was T-cell independent; both carrier-primed T helper cells and specific antigenic challenge, however, were required to trigger the localized B memory cells into antibody production. Specific B memory cell accumulation did not result from an expansion of the antigen-specific cell population due to local proliferation induced by antigen depots in the lymph nodes to challenge. Rather, the results indicated that recirculating B memory cells had progressively accumulated through retention by antigen in the lymph node. These findings suggest that, in the absence of T-cell help and specific antigenic challenge, B memory cells accumulate in lymphoid tissue (follicles) without responding and provide persistent local memory for the humoral immune response.