Regulatory properties of rabbit red blood cell hexokinase at conditions close to physiological

The true level of hexokinase in rabbit erythrocytes was determined by three different methods, including the spectrophotometric glucose-6-phosphate dehydrogenase coupled assay and a new radioisotopic assay. The value found at 37°C (pH 7.2) was 10.23±1.90 μmol/h per ml red blood cells, which is lower than previously reported values. More than 40 cellular components of the rabbit erythrocytes were tested for their effects on the enzyme. Their intracellular concentrations were also determined. Several of these compounds were found to be competitive inhibitors of the enzyme with respect to Mg·ATP^2?. Furthermore, reduced glutathione at a concentration of 1 mM was able to maintain hexokinase in the reduced state with full catalytic activity. The ability of orthophosphate to remove the inhibition of some phosphorylated compounds was examined under conditions similar to cellular (pH 7.2 and 50 μM of orthophosphate) and found to be of no practical interest. In contrast, the binding of ATP^4? and 2,3-diphosphoglycerate to the rabbit hemoglobin significantly modifies their intracellular concentrations and the formation of the respective Mg complexes. The pH-dependence of the reaction velocity and of the kinetic properties of the enzyme in different buffer systems were also considered. This information was computerized, and the rate of glucose phosphorylation in the presence of the mentioned compounds was determined. The value obtained, 1.94±0.02 μmol/h per ml red blood cells, is practically identical to the measured rate of glucose utilization by intact rabbit erythrocytes (1.92±0.3 μmol/h per ml red blood cells). These results provide further evidence for the central role of hexokinase in the regulation of red blood cell glycolysis.     

Rabbit red blood cell hexokinase

Summary Rabbit hexokinase (EC 2.7.1.1) has been shown to exist in reticulocytes as two distinct molecular forms, designated hexokinase Ia and Ib, but only one of these was consistently present in mature red cells. In vivo, hexokinase la and Ib show a decay rate of 3 and 8% a day, respectively, while in vitro they show a similar stability.The possibility that the proteolytic activities of the reticulocyte could be responsible for the fast decay of hexokinase was investigated. No differences were found in the decay rates of hexokinase la and Ib during in vitro reticulocyte maturation in presence or absence of proteolytic inhibitors. Contrariwise, many findings indicate the ATP-dependent proteolytic system of the reticulocyte as a possible mechanism. In fact, the decay of hexokinase and the degradation of 3H-globins are both stimulated by ATP and ubiquitin; they show similar kinetic properties and both disappear during reticulocyte maturation.The cellular localization of hexokinase la and Ib was shown to be responsible for the differences found between their decay rates.Abbreviations PMSF phenylmethylsulfonyl fluoride - TPCK 1-1-tosylamide-2-phenylethyl-chloromethyl ketone - TLCK N -p-tosyl-L-lysine chloromethyl ketone     

Histone mRNAs contain blocked and methylated 5′ terminal sequences but lack methylated nucleosides at internal positions

Histone mRNA, labeled with ³²P or ³H-methionine during the S phase of partially synchronized HeLa cells, was isolated from the polyribosomes and purified as a “9S” component by sucrose gradient sedimentation. We identified two types of 5′ terminals, m⁷G(5′)pppN^mpN and m⁷G(5′)pppN^m-pN^mpN, in which the first methylated nucleoside is 7-methylguanosine, the second is either N⁶,2′-O-dimethyladenosine, 2′-O-methyladenosine, or 2′-O-methylguanosine, and the third is 2′-O-methyluridine, 2′-O-methylcytidine, or 2′-O-methyladenosine. Approximately 1.7% of the ³²P label was present in the 5′ terminal structures. Assuming a similar specific radioactivity for all phosphates, this percentage corresponds to an average of one terminal per 335 nucleotides. Histone mRNA differed from bulk polyadenylylated mRNA of HeLa cells in lacking significant amounts of 2′-O-methyluridine or 2′-O-methylcytidine in the second position of the 5′ terminal oligonucleotide and in lacking N⁶-methyladenosine residues at internal positions.     

Monoclonal antibodies directed against the sugar sequence of lacto-N-fucopentaose III are obtained from mice immunized with human tumors

Four hybridomas obtained from mice immunized with human adenocarcinomas of colon or stomach produce antibodies that bind specifically in solid-phase radioimmunoassay to the ceramide pentasaccharide that contains the lacto-N-fucopentaose III sequence of sugars. Binding of the antibodies to the glycolipid is inhibited by lacto-N-fucopentaose III,

but not by structurally related oligosaccharides. The antibodies bind to glycolipids of erythrocytes, granulocytes, and certain normal and malignant tissues.     

Evidence for a particulate location of ubiquitin conjugates and ubiquitin-conjugating enzymes in rabbit brain.

Conjugate ubiquitin was previously found in the nucleus, cytoplasm, and membranes of eukaryotic cells while the enzymes of the ubiquitin-conjugating system appear to be cytoplasmic. We have prepared the mitochondrial fraction from rabbit brain by discontinuous density gradient ultracentrifugation and by Western blotting, using a specific antibody against conjugate ubiquitin, showing that it contains ubiquitin conjugates in a very wide molecular weight range. Electron microscopy and measurement of specific enzyme markers show that this fraction not only contains mitochondria but also some endoplasmic reticulum vesicles. Immunostaining with anti-ubiquitin IgG followed by immunodecoration with colloidal gold particles provides evidence for the presence of conjugate ubiquitin both in mitochondria and in the endoplasmic reticulum. Furthermore, this "mitochondrial fraction" shows a pronounced ATP-dependent ability to conjugate 125I-ubiquitin into a number of endogenous proteins as evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Addition of E1, E2, and E3, the enzymes of the ubiquitin conjugating system purified from rabbit reticulocytes, does not further increase this ubiquitination nor incorporate 125I-ubiquitin into additional protein bands. The same mitochondrial fraction is not able to carry out any ATP-dependent degradation of 125I-albumin; however, it contains an isopeptidase activity able to release the covalently incorporated 125I-ubiquitin and is also able to conjugate 125I-ubiquitin to exogenous proteins as oxidized RNase. By affinity chromatography on ubiquitin-agarose of fraction II of a crude Triton X-100 extract of the mitochondrial fraction, several proteins corresponding in Mr to the E1 and E2s enzymes were obtained. These proteins were also able to form specific ubiquitin-thiol ester bounds on sodium dodecyl sulfate-polyacrylamide gels and to support 125I-ubiquitin conjugation to oxidized RNase. Detergent fractionation of the mitochondrial fraction provided evidence for a possible localization of the ubiquitin conjugating activity in the mitochondrial external membrane and endoplasmic reticulum. The presence of an active ubiquitin protein conjugating system in mitochondria and endoplasmic reticulum may be related to the turnover of organelle proteins as well as to specific cell functions such as import of proteins into mitochondria and ubiquitination of externally oriented membrane-bound proteins.     

Glycolipids of cell surfaces: Isolation of specific sugar sequences using carbohydrate-binding proteins

Proteins that bind carbohydrates can be used to isolate specific sugar sequences from complex mixtures. Free sialyloligosaccharides or sialyloligosaccharides released from gangliosides by ozonolysis and alkaline fragmentation are labeled at their reducing ends by reduction with NaB[³H]₄. After partial separation by column chromatography, oligosaccharide fractions are tested for binding to anti-sialyloligosaccharide antibodies [Smith, D. F., and Ginsburg, V. (1980) J. Biol. Chem.255, 55–59] and cholera toxin by a nitrocellulose filter assay. Oligosaccharides bound by the proteins can be eluted from the filters and further characterized. The method can be used to isolate and identify carbohydrate ligands of cell surfaces.     

Cynanchoside,a highly oxygenated iridoid glucoside from Macfadyena cynanchoides

The elucidation of the structure and stereochemistry of cynanchoside, a new highly oxygenated iridoid glucoside isolated from Macfadyena cynanchoides (Bignoniaceae), has been accomplished using mainly ¹H and ¹³C NMR spectral data and further confirmed by simple chemical transformations.     

Photosensory transduction in Euglena gracilis: Effect of some metabolic drugs on the photophobic response

We have investigated the effect of some metabolic drugs, 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), 2,4-dinitrophenol (DNP), sodium azide (NaN₃), on the photobehavior of single cells of Euglena gracilis, in order to clarify the relevance of different metabolic pathways in the process of photoperception and sensory transduction in this alga. The results obtained show that the photophobic response of Euglena is not affected by the action of these drugs. This suggests that neither the photosynthetic process nor oxidative phosphorylation play a significant role in the phenomenon of photosensory transduction in Euglena.List of Abbreviations DNP 2,4-dinitrophenol - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - PSI Photosystem I - PSII Photosystem II     

In vitro targeting of erythrocytes to cytotoxic T-cells by coupling of Thy-1.2 monoclonal antibody.

A method was explored to develop a general means to target erythrocytes to T-cells in vitro. Mouse erythrocytes were coupled with an anti-Thy-1.2 monoclonal antibody by two methods. Chromic chloride coupling of antibody was preferred to biotinylation. The morphology, osmotic fragility, and the hematological values of treated cells were normal compared with those of control erythrocytes. Antibody-coupled erythrocytes were incubated with cytotoxic T-lymphocytes (CTLL) in vitro at a 20:1 ratio. Approximately 60-70% of the CTLL formed rosettes. The cell mixture was subjected to gradient centrifugation and separated into four fractions. THe rosettes were clearly identified only in the treated group containing anti-Thy-1.2-coupled erythrocytes. No rosettes were found when aspecific monoclonal antibody was coupled to erythrocytes. Examination by scanning and transmission electron microscopy revealed CTLL with 4-5 erythrocytes attached to them but did not show any evidence of membrane fusion. Rosettes of CTLL incubated in vitro proliferated as well as CTLL alone and maintained their dependency on interleukin-2. Targeting of erythrocyte carriers to lymphocytes offers the potential for delivery of molecules directly to the target cell.