Compartmentation of hexokinase in human blood cells characterization of soluble and particulate enzymes

The isozyme distribution, kinetic properties and intracellular localization of hexokinase (ADP: D-hexose-6-phosphotransferase, EC 2.7.1.1) were studied in erythrocytes, blood platelets, lymphocytes and granulocytes. Soluble and particulate fractions were separated by a rapid density centrifugation method after controlled digitonin-induced cell lysis. In lymphocytes and platelets the major part of total activity was particle-bound (78 and 88%, respectively). In granulocytes and erythrocytes most of the hexokinase activity was found in the cytosol. All cell types, except granulocytes, contain mainly the type I isozyme. Platelets contain only type I hexokinase, while in lyphocytes a minor amount of type III is present in the soluble fraction (less than 10% of total activity). The major constituent of granulocytes is type III hexokinase (70–80% of total activity), the remaining 20–30% is type I hexokinase. Erythrocytes contain a multibanded type I hexokinase. The substrate affinities of the type I hexokinase do not differ significantly between the different cell types or between soluble, bound and solubilized fractions. Only soluble hexokinase from lymphocytes shows a slightly decreased K_m apparent for glucose (P < 0.05).     

Cultured endothelial cells produce heparinlike inhibitor of smooth muscle cell growth

Using cultured cells from bovine and rat aortas, we have examined the possibility that endothelial cells might regulate the growth of vascular smooth muscle cells. Conditioned medium from confluent bovine aortic endothelial cells inhibited the proliferation of growth-arrested smooth muscle cells. Conditioned medium from exponential endothelial cells, and from exponential or confluent smooth muscle cells and fibroblasts, did not inhibit smooth muscle cell growth. Conditioned medium from confluent endothelial cells did not inhibit the growth of endothelial cells or fibroblasts. In addition to the apparent specificity of both the producer and target cell, the inhibitory activity was heat stable and not affected by proteases. It was sensitive flavobacterium heparinase but not to hyaluronidase or chondroitin sulfate ABC lyase. It thus appears to be a heparinlike substance. Two other lines of evidence support this conclusion. First, a crude isolate of glycosaminoglycans (TCA-soluble, ethanol-precipitable material) from endothelial cell-conditioned medium reconstituted in 20 percent serum inhibited smooth muscle cell growth; glycosaminoglycans isolated from unconditioned medium (i.e., 0.4 percent serum) had no effect on smooth muscle cell growth. No inhibition was seen if the glycosaminoglycan preparation was treated with heparinase. Second, exogenous heparin, heparin sulfate, chondroitin sulfate B (dermatan sulfate), chondroitin sulfate ABC, and hyaluronic acid were added to 20 percent serum and tested for their ability to inhibit smooth muscle cell growth. Heparin inhibited growth at concentrations as low as 10 ng/ml. Other glycosaminoglycans had no effect at doses up to 10 μg/ml. Anticoagulant and non- anticoagulant heparin were equally effective at inhibiting smooth muscle cell growth, as they were in vivo following endothelial injury (Clowes and Karnovsk. Nature (Lond.). 265:625-626, 1977; Guyton et al. Circ. Res. 46:625-634, 1980), and in vitro following exposure of smooth muscle cells to platelet extract (Hoover et al. Circ. Res. 47:578-583, 1980). We suggest that vascular endothelial cells may secrete a heparinlike substance in vivo which may regulate the growth of underlying smooth muscle cells.     

LIGHT ACTION SPECTRA OF N2 FIXATION BY HETEROCYSTOUS CYANOBACTERIA FROM THE BALTIC SEA1

An on‐line, laser photo‐acoustic, trace gas detection system in combination with a stepper motor‐controlled monochromator was used to record semicontinuous light action spectra of nitrogenase activity in heterocystous cyanobacteria. Action spectra were made of cultures of Nodularia spumigena Mertens ex Bornet & Flahault, Aphanizomenon flos‐aquae Ralfs ex Bornet & Flahault, and Anabaena sp. and from field samples of a cyanobacterial bloom in the Baltic Sea. Nitrogenase activity was stimulated by monochromatic light coinciding the red and blue peaks of chl a, the phycobiliproteins phycocyanin (allophycocyanin) and phycoerythrin, and several carotenoids. Because nitrogenase is confined to the heterocyst, it was concluded that all photopigments must have been present in these cells, were involved in light harvesting and photosynthesis, and supplied the energy for N₂ fixation. The species investigated showed marked differences in their nitrogenase action spectra, which might be related to their specific niches and to their success in cyanobacterial blooms. Moreover, light action spectra of nitrogenase activity shifted during the day, probably as the result of changes in the phycobiliprotein content of the heterocyst relative to chl a. Action spectra of nitrogenase and changes in pigment composition are essential for the understanding of the competitive abilities of species and for the estimation of N₂ fixation by a bloom of heterocystous cyanobacteria.     

Substructure of the glomerular slit diaphragm in freeze-fractured normal rat kidney

In the renal glomerulus, the narrow slits between adjacent epithelial podocytes are bridged by a diaphragm (2, 8, 11). In rat and mouse kidneys fixed by perfusion with tannic acid and glutaraldehyde (TAG), it has recently been discovered that this diaphragm has a highly ordered, isoporous substructure (9). It consists of a regular array of alternating cross bridges extending from the podocyte plasma membranes to a centrally running filament. This zipperlike pattern results in two rows of rectangular pores, approximately 40 X 140 A in cross section, dimensions consistent with the proposed role of the diaphragm as an important filtration barrier to plasma proteins (6). In the present study, we found in freeze-cleaved and in freeze-etched normal rat glomeruli that the surface of the slit diaphragm has an appearance conforming to the pattern found in sectioned material.     

Phenotypes of pleiotropic-negative sporulation mutants of Bacillus subtilis

The phenotypic properties of representatives of the five genetic classes of pleiotropic-negative sporulation mutants have been investigated. Protease production, alkaline and neutral proteases, was curtailed in spoA mutants, but the remainder of mutant classes produced both proteases, albeit at reduced levels. The spoA and spoB mutants plaqued phi2 and phi15 at high efficiency, but the efficiency of plating of these phages on spoE, spoF, and spoH mutants was drastically reduced. Antibiotic was produced by the spoH mutants and to a degree by some spoF mutants, but the other classes did not produce detectable activity. The spoA mutants were less responsive to catabolite repression of histidase synthesis by glucose than was the wild type. Severe catabolite repression could be induced in spoA mutants by amino acid limitation, suggesting that the relaxation of catabolite repression observed is not due to a defect in the mechanism of catabolite repression. Although others have shown a perturbation in cytochrome regulation in spoA and spoB mutants, the primary dehydrogenases, succinate dehydrogenase and reduced nicotinamide adenine dinucleotide dehydrogenase, leading to these cytochromes are unimpaired in all mutant classes. A comparison of the structural components of cell walls and membranes of spoA and the wild type is made. The pleiotropic phenotypes of these mutants are discussed.     

Wing development and parthenogenesis induced in progenies of kinoprene-treated gynoparae of Aphis fabae and Myzus persicae

Presumptive gynoparae of Aphis fabae and Myzus persicae were exposed to various levels of kinoprene (Zoecon's ZR 777) by being placed as 4th-instar alatiform larvae on bean or radish seedlings that had been sprayed with different concentrations of kinoprene in an acetone-tween-water emulsion. Larvae exposed to the highest (0.1%) concentration tested developed into adults 1 to 2 days sooner than those on control plants. The adults on the treated plants had variously deformed wings, reduced sclerotization (and pigmentation in the case of M. persicae) and other apteriform features. On reaching adulthood the affected aphids settled to feed and started to larviposit some days earlier than the control aphids. After two weeks as adults, treated gynoparae of M. persicae produced more larvae than the 7 to 9 typically deposited by control gynoparae under the short-day and cool temperature conditions employed in these tests.Whereas most or all of the larvae produced by the control gynoparae developed into oviparae (apterous, egg-laying, sexual females), gynoparae exposed to 0.1% kinoprene-treated plants predominantly produced alatiform viviparous offspring. If the latter were allowed to develop on untreated plants they deposited a few oviparous larvae. Alatiform virginoparae of M. persicae (from the same holocyclic strain that produced the gynoparae) also responded to kinoprene by developing wing deformities and by producing alatiform offspring. In contrast, alatiform virginoparae from an androcyclic strain of M. persicae, although developing wing deformities, produced only apterous progeny.The stimulation by kinoprene of wing development and parthenogenesis in the progeny of treated gynoparae is discussed in the light of our present knowledge of these aspects of aphid polymorphism.     

Isoenzymes of phosphofructokinase in the rat. Demonstration of the three non-identical subunits by biochemical, immunochemical and kinetic studies.

In man and the rabbit, 6-phosphofructokinase (PFK, EC 2.7.1.11) exists in tetrameric isoenzymic forms composed of muscle (M or A), liver (L or B) and platelet or brain (P or C) subunits, which are under separate genetic control. In contrast, the genetic control of the rat PFK has not yet been conclusively established; it is unclear whether the P-type or C-type subunit exists in this species. To resolve this question, we investigated the enzyme from the skeletal muscle, liver and brain of rats of Wag/Rij strain. Our studies demonstrate that the rat PFK is also under the control of three structural loci and that the homotetramers M4, P4 and L4 exhibit unique chromatographic, immunological and kinetic-regulatory properties. Skeletal-muscle and brain PFKs consist of isolated M4 and P4 homotetramers respectively. Although liver PFK consists predominantly of L4 homotetramer, it also contains small amounts of PL3 and P2L2 species. All three PFKs exhibit allosteric properties: co-operativity with fructose 6-phosphate and inhibition by ATP decrease in the order P4 greater than L4 greater than M4. P4 and M4 tetramers are the most sensitive to citrate inhibition, whereas L4 tetramer is the least sensitive. More importantly, P4 and L4 isoenzymes are the most sensitive to activation by fructose 2,6-bisphosphate, whereas M4 isoenzyme is the least sensitive. These results indicate that the brain PFK in this strain of rat is a unique tetramer, P4, which also exhibits allosteric kinetics, as do the well-studied M4 and L4 isoenzymes. The reported differences in the number and nature of isoenzymes present in the rat brain and liver most probably reflect the differences in the strains studied by previous investigators. Since the nature of the rat PFK isoenzymes and nomenclatures reported by previous investigators have been now reconciled, it is proposed that, for the sake of uniformity, only well-established nomenclatures used for the rabbit or human PFK isoenzymes be used for the rat isoenzymes.     

Activity and isoenzyme patterns of glycolytic enzymes during perinatal development of rat lung

The enzymes hexokinase (EC 2.7.1.1), phosphofructokinase (EC 2.7.1.11), enolase (EC 4.2.1.11) and pyruvate kinase (EC 2.7.1.40) were studied in rat lung during development starting at day 16 of gestation (day-6) until 5 days after birth. During gestation, the activities of hexokinase type II, enolase and pyruvate kinase decreased and reached adult values at birth or shortly thereafter. Hexokinase type I remained relatively constant and the decrease of soluble type II hexokinase was compensated for by an increment of particle-bound hexokinase starting at day 20 of gestation until birth. In contrast, phosphofructokinase activity increased until day 20 of gestation followed by a rapid fall in activity until 2 days after birth. Except for hexokinase no isoenzyme shifts were observed in the period of observation. The results are discussed with respect to the proposed relationship between glycogen breakdown and surfactant synthesis during the perinatal period and suggest a regulatory role for phosphofructokinase in this process.     

Author index for volume 31

Kilham rat virus (KRV) was isolated from the lymphocyte cytopathic 13762 summary adenocarcinoma tumor line described in part I of this report, as well as three other in vivo passaged rat tumors maintained in the same animal room. It could not, however, be isolated from the noncytopathic (CS8NT)D tissue culture line. Tests done with CsCl₂-purified KRV preparations showed that the virus could replicate in rat lymphocytes and could profoundly depress lymphocyte viability and lymphoproliferative responses. Heterologous anti-KRV antiserum could reverse the inhibitory effects of the purified virus preparation and the inhibitory effects of ultrasonically disrupted KRV-infected tumor cells, but could only partially reverse the inhibitory properties of X-irradiated whole 13762 tumor cells. The results suggest that KRV could account for some, if not all, of the inhibitory properties of the 13762 tumor line.