Glycogen phosphorylase from human leukocytes. Isolation and kinetic properties

Homogeneous glycogen phosphorylase from human leukocytes has been obtained. A one-step bioluminescent procedure for the enzyme activity assay has been developed. This method is based on a continuous recording of the product of the glycogen phosphorylase-catalyzed reaction using a coimmobilized multienzyme system (phosphoglucomutase, glucose-6-phosphate dehydrogenase, NADH:FMN oxidoreductase and bacterial luciferase). The method sensitivity is 10 times as high compared to earlier described methods. The Km values for glycogen (0.2 mg/ml) and phosphate (3.9 mM) at pH 7.9 were determined. AMP was shown to be the enzyme effector.     

Isolation from human chronic-lymphocytic-leukaemia cells of membrane glycoproteins associated with Fc-receptor functions. Physical parameters and production of polyclonal antibodies.

Two membrane glycoproteins that bound immune complexes and inhibited Fc-receptor- (FcR-)mediated functions in vitro were purified from human FcR+ chronic-lymphocytic-leukaemia cells. A multi-step purification was developed, consisting essentially in: (i) Tween 40 extraction of crude cell membranes; (ii) solubilization of membrane fragments by Renex-30; (iii) isolation of glycoproteins by affinity chromatography on Lens culinaris haemagglutinin-Sepharose; (iv) papain treatment of the eluted glycoproteins followed by gel-filtration chromatography; (v) purification by polyacrylamide-gel electrophoresis of two molecular species from the protein-size fraction enriched for immune-complex-binding activity. The two electrophoretically isolated components displayed apparent molecular masses of 70 and 45 kDa by SDS/polyacrylamide-gel electrophoresis and restricted charge heterogeneity by two-dimensional analysis. Two-dimensional peptide mapping revealed the presence of many peptides in common between the two proteins and the absence of a number of peptides in the 45 kDa component. These two polypeptides were used as immunogens to produce polyclonal antibodies that cross-reacted with both proteins and specifically inhibited FcR-mediated reactions in vitro. Furthermore, FcR-related components from detergent-extracted lysates of the human K562 and U937 cell lines or human placental membranes were revealed by the putative anti-FcR antibodies adsorbed on Protein A-Sepharose.     

Isolation and properties of alpha-D-mannosidase from human kidney.

Alpha-D-Mannosidase activity exists in three forms that can be separated by DEAE-cellulose chromatography, alpha-D-Mannosidase was isolated from human kidney in a homogeneous state, and was purified 2100-fold, with p-nitrophenyl alpha-D-mannoside as substrate. The purified alpha-D-mannosidase was practically free from all other glycosidases tested. The Km of the synthetic substrate with the enzyme was 1 X 10(-3) M and the pH optimum 4.5. It was inhibited by heavy metals, sodium dodecyl sulphate, urea and compounds that react with the thiol groups, and was activated by Zn2+, Na+, 2-mercaptoethanol, human albumin and gamma-globulin. The mol. wt. of the enzyme was estimated to be 180 000 +/- 4500. After pretreatment with 2-mercaptoethanol and sodium dodecyl sulphate, alpha-D-mannosidase dissociated into subunits of mol. wts. of 58 000 +/- 600 and 30 000 +/- 380 respectively. Subunits of the same molecular weights were also obtained after the enzyme was heated at 100 degrees C.     

Passive mechanical properties of human leukocytes.

Micropipette experiments are used to determine the rheological properties of human leukocytes. Individual cells in EDTA are subjected to a known aspiration pressure via a micropipette, and their surface deformation from the undeformed spherical shape is recorded on a television monitor. The cells are mathematically modeled as homogeneous spheres, and a standard solid viscoelastic model is found to describe accurately the deformation of the cell for small strains. These experimental and theoretical studies provide the basis for further investigations of leukocyte rheology in health and disease.     

Crystallization and properties of myeloperoxidase from normal human leukocytes

Myeloperoxidase was purified from normal human leukocytes in a crystalline state. Two types of crystals were obtained by the batchwise and dialysis crystallization methods, one of which had a bipyramidal shape belonging to the orthorhombic system. Three multiple forms of human myeloperoxidase were separated from the crystalline enzyme by CM-Sepharose chromatography with sodium chloride gradient elution. These three multiple forms were found to have very similar enzymatic, spectroscopic, and chemical properties. However, slight differences were observed in their amino acid compositions and the molecular weights of their large subunits determined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Hemi-myeloperoxidase was prepared from holo-myeloperoxidase by reduction with dithiothreitol and modification with iodoacetamide, and the molecular shapes of the holo- and hemi-enzymes were determined by analytical ultracentrifugation. The axial ratios were calculated to be 2.4-3.5 for the holo-enzyme and 2.9-3.1 for the hemi-enzyme. These results suggest that the shapes of the two enzymes are more spherical in solution than the proposed structural model previously reported.     

Antiidiotypic antibodies against anti-cGMP polyclonal antibodies.

Affinity-purified polyclonal anti-cGMP antibodies were obtained from rabbit serum after immunization by succinyl derivative of cGMP coupled to bovine serum albumin. These antibodies were used to raise antiidiotypic antibodies in rats. Putative antiidiotypic serum inhibited the binding of [3H]cGMP to affinity-purified anti-cGMP antibodies. The influence of immunoglobulins isolated from antiidiotypic serum on the ion conductance of rod outer segment plasma membrane fragments from frog retina was studied in patch-clamp experiments. These immunoglobulins increased the conductance of ion channels acting like a natural agonist (cGMP). Preimmune immunoglobulins did not act. The data obtained suggest that antiidiotypic antibodies interact with regulatory cGMP-binding sites of the plasma membrane channels.     

An automatic multidimensional chromatography system for purification of human uterine progesterone receptor and induction of polyclonal antibodies

This paper reports on the synthesis of Org2058-bonded microparticulate silicas and their use in affinity chromatography as the first step for the purification of human progesterone receptor. The development of microprocessor-controlled instruments allows all the various steps to be performed automatically. The various steps used for the purification of human progesterone receptor were carried out with the FPLC system: affinity chromatography, desalting of eluate on Sephadex G-25, anion-exchange chromatography using a Mono Q column. With this procedure the receptor was purified approx. 10,000-fold within 24 h. The yield of receptor was generally 85-95%. Investigations with induced anti-progesterone receptor antibodies obtained after the fourth immunization show their immunoreactive behaviour towards progesterone receptor in crude cytosol, which was proved by sucrose density gradient centrifugation and by gel filtration on the FPLC system using a Sepharose 12 column. This implies that progesterone receptor was efficiently purified by our purification procedure.     

Isolation of human salivary polymorphonuclear leukocytes and their stimulation-coupled responses.

A simple method was developed to isolate viable human salivary polymorphonuclear leukocytes (SPMN) from the oral cavity, and stimulation-coupled responses of these cells were examined. From morphological characteristics and the presence of neutrophil-specific annexin protein (39-kDa protein), we found that these cells seemed to be very similar to human peripheral polymorphonuclear leukocytes (PPMN), although they were in rather young stages. Stimulation-coupled responses of these cells were observed in terms of superoxide (O2.-) genration, luminol chemiluminescence response (LCL), membrane depolarization, and changes in intracellular calcium ion concentration ([Ca2+]i). The rates of superoxide generation by various stimuli, such as formylmethionylleucylphenylalanine (FMLP), phorbol 12-myristate 13-acetate (PMA) and opsonized zymosan (OZ) were different. Superoxide generation and strong chemiluminescence response were observed without addition of any stimuli. This endogenous LCL was inhibited by azide and superoxide dismutase (SOD), but not by uric acid (UA). The intensity of the endogenous LCL decreased with time after isolation from the oral cavity. This decrease was accompanied by the appearance of a FMLP-coupled response. Furthermore, the endogenous activity which produced active oxygen species was maintained in the medium at 4 degrees C for a long period after isolation. From these results, it is suggested that SPMN have the ability to show characteristic responses to various stimuli, and that SPMN play important roles in the defense mechanisms in the oral cavity.     

Kininase II from human seminal plasma. Isolation and properties

A rapid and highly efficient procedure for purification of kininase II from human seminal plasma is described. After ultra centrifugation, the enzyme was purified by gel filtration on Sepharose 6B CL and ion exchange chromatography followed by affinity chromatography of EDTA-inhibited enzyme on bradykinin-Sepharose. The enzyme was specifically inhibited by Captopril and BPP9a but not by phosphoramidon. PAGE in the presence of sodium dodecyl sulfate under reducing conditions resulted in two major protein bands with apparent molecular masses of about 55 kDa and 65 kDa and two faint protein bands at higher molecular masses. Antibodies raised against the major protein bands showed full cross reactivity with all four protein bands. The presented data indicate that kininase II consists of subunits.     

Purification and properties of alkaline phosphatase from human polymorphonuclear leukocytes

A procedure for the purification of alkaline phosphatase from human polymorphonuclear leukocytes is described, involving enzyme solubilisation with Triton X-100 and chromatography on DEAE-Sepharose CL 6B and Cibacron Red F = B-Sepharose 4B. The final enzyme preparation was 244-fold purified and was shown to be capable of hydrolysis of a wide range of phosphorylated substates.     

Isolation of three native forms of myeloperoxidase from human polymorphonuclear leukocytes

The bacteriophage phi X174 gene A encodes two proteins: gene A protein and A* protein. Purified A* protein acts as a single-stranded, DNA-specific endonuclease which remains covalently attached to the 5'-end of the cleavage site. Incubation of A* protein with the synthetic heptamer CAACTTG or with oligonucleotides which yield this heptamer after cleavage with the A* protein yields oligonucleotides with the sequences CAACTTGAG, CAACTTGAGG and CAACTTGAGGA. This indicates that A* protein carries an oligonucleotide with the sequence--AG, -AGG or -AGGA. The oligonucleotide can be transferred to the 3'-end of the heptamer CAACTTG. This suggests that A* protein reacts with a specific DNA sequence in the infected cell.     

Isolation procedure and some properties of myeloperoxidase from human leucocytes.

1. A rapid isolation procedure with a high yield for pure myeloperoxidase (donor:H2O2 oxidoreductase, EC 1.11.1.7) from normal human leucocytes is described. The enzyme was solubilized from leucocytes with the detergent, cetyltrimethylammonium bromide, and purified to apparent homogeneity. The yield of the enzyme was 17% with an absorbance ratio A430nm/A280nm = 0.85. 2. The purified enzyme showed three isoenzyme bands after polyacrylamide gel electrophoresis; ultracentrifuge studies indicated one homogeneous band with a molecular weight of 144 000. After reduction of myeloperoxidase, sodium dodecyl sulfate gel electrophoresis resolved an intense band (63 000 daltons) and a weak band (81 000 daltons). 3. The carbohydrate content of the enzyme was at least 2.5%. Mannose, glucose and N-acetylglucosamine were present. The amino acid composition is reported. 4. The EPR spectrum exhibited a high-spin heme signal with rhombic symmetry (gx = 6.92, gy = 5.07 and gz = 1.95). Upon acidification this signal was converted into a signal with more axial symmetry (g perpendicular = 5.89). At high pH (9.5) the EPR spectrum of the enzyme only shows low-spin ferric heme resonances. The circular dichroism spectra of ferric and ferrous myeloperoxidase in the visible and ultraviolet region show maxima and minima in ellipticity.     

Mechanism for induction of anti-DNA antibodies by bacterial lipopolysaccharides in mice; II. Correlation between anti-DNA induction and polyclonal antibody formation by various polyclonal B lymphocyte activators.

The capacity of various polyclonal B lymphocyte activators (PBA) to induce, in mice, the formation of anti-DNA antibodies was compared with their ability to mediate the release of DNA in circulating blood and to stimulate polyclonal antibody synthesis in vivo. Anti-DNA antibodies or polyclonal antibody synthesis were induced in mice after the injection of at least 10 microgram lipopolysaccaride (LPS) from Salmonella typhimurium, 1 mg dextran sulfate (DS), or 2 mg purified protein derivative of tubercle bacteria RT32 (PPD). Smaller quantities of LPS (0.1 microgram) or DS (500 microgram) were sufficient to cause the release of DNA in circulating blood, whereas PPD was not able to provoke such a release at any concentration used. The association of anti-DNA antibodies with polyclonal antibody synthesis in mice injected with various PBA contrasts with the lack of correlation between the formation of anti-DNA antibodies and the release of measurable amounts of DNA in circulating blood. These results strongly suggest that the induction of anti-DNA antibodies by PBA is a consequence of the polyclonal B lymphocyte activation.     

Isolation of high affinity human antibodies directly from large synthetic repertoires.

Antibody fragments of moderate affinity (approximately microM) can be isolated from repertoires of approximately 10(8) immunoglobulin genes by phage display and rounds of selection with antigen, and the affinities improved by further rounds of mutation and selection. Here, as an alternative strategy, we attempted to isolate high affinity human antibodies directly from large repertoires. We first created highly diverse repertoires of heavy and light chains entirely in vitro from a bank of human V gene segments and then, by recombination of the repertoires in bacteria, generated a large (close to 6.5 x 10(10)) synthetic repertoire of Fab fragments displayed on filamentous phage. From this repertoire we isolated Fab fragments which bound to a range of different antigens and haptens, and with affinities comparable with those of antibodies from a secondary immune response in mice (up to 4 nM). Although the VH-26 (DP-47) segment was the most commonly used segment in both artificial and natural repertoires, there were also major differences in the pattern of segment usage. Such comparisons may help dissect the contributions of biological mechanisms and structural features governing V gene usage in vivo.