Separation of two PZ-peptidases from bovine dental follicle.

Two PZ-peptidases (EC 3.4.-) (A and B) cleaving a synthetic substrate for collagenase, 4-phenylazobenzyloxycarbonyl-L-Pro-L-Leu-Gly-L-Pro-D-Arg (PZ-peptide) have been separated from the particulate fraction of bovine dental follicle. PZ-peptidase A had a molecular weight of 220 000, an optimum pH at 8.0-8.5, and a Km value of 67 muM toward PZ-peptide at pH 7.1, whereas PZ-peptidase B had a molecular weight of 20 000, an optimum pH at 6.5-6.7, and a Km value of 400 muM toward PZ-peptide at pH 7.1. Two similar enzymes were also isolated from the soluble fraction. Since the pH-activity curve of the crude tissue preparations such as homogenate, microsomes and soluble supernatant had two peaks at 6.5-6.7 and 8.0-8.5, both PZ-peptidase A and B may exist in situ as two independent active enzymes.     

Purification and properties of a peptidase acting on a synthetic substrate for collagenase from monkey kidney.

A peptidase cleaving a synthetic substrate for collagenase, 4-phenylazobenzyloxycarbonyl-L-Pro-L-Leu-Gly-L-Pro-D-Arg (designated as PZ-peptide) has been purified extensively (about 5200-fold) from a soluble extract of monkey kidney with a view of carrying out studies on its possible physiological role. The purified PZ-peptidase appeared essentially free of collagenase, nonspecific protease and di- and tri-peptidase activities. The properties of the purified PZ-peptidase resemble very much the granuloma enzyme. It is optimally active around pH 7.0. Its apparent Km value for PZ-peptide is 0.72 mM and V is 10.1 mumol/mg protein/min. It is reversibly inhibited by p-hydroxymercuribenzoate and HgCl2, whereas iodoactetamide does not affect the enzyme activity. N-Ethylmaleimide inhibited the enzyme partially (50%). Heavy metals like Cu-2+, Cd-2+, Ag+, Pb-2+, Ni-2+, and Zn-2+ completely inhibited the enzyme activity, while the inhibition by Co-2+ was only partial. Fe-2+ did not exert any effect on the activity. The enzyme activity was completely inhibited by EDTA and was restored almost to the original value by metal ions like Mn-2+, Mg-2+, Ca-2+ and Ba-2+. The approximate molecular weight of the purified enzyme was estimated to be 56 000.     

Subcellular distribution of glucocorticoid receptors in mouse fibroblasts.

Mouse fibroblasts contain a macromolecular binding component (receptor) which binds glucocorticoids specifically and with high affinity. This study shows that there are three different cellular forms of bound receptor and that it is experimentally possible to markedly alter the subcellular distribution of these three forms. Cells incubated with (3H)triamcinolone acetonide were broken after hypotonic shock and a 7000g hypotonic supernatant was obtained; the pellet was extracted with 0.3 M KCl, yielding a nuclear extract; the remaining pellet was resuspended in water, sonicated, and assayed for "nuclear residual" (i.e., nonextractable) radioactivity. If whole cells are incubated at 0 degrees in a growth medium, almost all of the bound steroid is located in the hypotonic supernatant fraction. Incubation at 37 degrees produces a shift of the steroid-bound macromolecule into the nuclear extractable form, while omission of glucose and addition of KCN at 37 degrees markedly increase the nuclear residual form at the expense of both the nuclear-extractable and supernatant forms. Since DNase treatment of chromatin liberates a soluble steroid-receptor complex, we believe that the nuclear residual form may be steroid-receptor complex tightly bound to chromatin. We propose a model suggesting that an energy-requiring process is required to generate free receptor from the chromatin complex to complete the normal cellular recycling system.     

Axonal transport of proteoglycans to the goldfish optic tectum

The study addressed the question of whether³⁵SO₄ labeled molecules that the have been delivered to the goldfish optic nerve terminals by rapid axonal transport include soluble proteoglycans. For analysis, tectal homogenates were subfractionated into a souluble fraction (soluble after centrifugation at 105,000g), a lysis fraction (soluble after treatment with hypotonic buffer followed by centrifugation at 105,000g) and a final 105,000g pellet fraction. The soluble fraction contained 25.7% of incorporated radioactivity and upon DEAE chromatographys was resolved into a fraction of sulfated glycoproteins eluting at 0–0.32 M NaCl and containing 39.5% of total soluble label and a fraction eluting at 0.32–0.60 M NaCl containing 53.9% of soluble label. This latter fraction was included on columns of Sepharose CL-6B with or without 4 M guanidine and after pronase digestion was found to have 51% of its radioactivity contained in the glycosaminoglycans (GAGs) heparan sulfate and chondroitin (4 or 6) sulfate in the ratio of 70% to 30%. Mobility of both intact proteoglycans and constituent GAGs on Sepharose CL-6B indicated a size distribution that is smaller than has been observed for proteoglycans and GAGs from cultured neuronal cell lines. Similar analysis of lysis fraction, containing 11.5% of incorporated³⁵SO₄, showed a mixture of heparan sulfate and chondroitin sulfate containing proteoglycans, apparent free heparan sulfate and few, if any, sulfated glycoproteins. Overall, the result support the hypothesis that soluble proteoglycans are among the molecules axonally transported in the visual system.     

Purification of surface membranes from immature brain cells

A procedure is described for the isolation of surface membranes from immature neurons. The cells are swollen in a hypotonic medium, homogenized, and membranes isolated by gradient centrifugation. The crude membrane fraction contains large vesicles and axon-like sacs. It represents 3 % of the homogenate protein. After lysis it produces a soluble extract and purified surface membranes. The extract is characterized by a high concentration of microtubular protein. Surface membranes show a high activity of Na⁺-K⁺-activated ATPase. The labelling of proteins in surface membranes in vivo is characterized by a delayed accumulation of radioactivity.     

Adenosine 3′:5′-monophosphate-dependent an independent histone kinases isolated from human tonsillar lymphocytes

Human tonsillar lymphocytes were fractionated by a hypotonic extraction procedure, followed by the purification and extraction of nuclei. Two different histone kinases could be separated by DEAE-cellulose chromatography from the hypotonic extract, each of which phosphorylated the F_2b histone fraction preferentially. In the nuclear extract, a third histone kinase fraction was found, which primarily phosphorylated the F_2a histone. Only one of the histone kinases, found in the hypotonic extract, was cyclic AMP dependent, and the other two enzymes were independent of the cyclic nucleotide. In contrast with the other two histone kinases the cyclic AMP-dependent enzyme had a specific effect, phosphorylating mainly one particular site of the F_2b histone fraction in the presence of cyclic AMP.     

Latent acetylcholinesterase in secretory vesicles isolated from adrenal medulla

A new procedure is described for the preparation of highly purified and stable secretory vesicles from adrenal medulla. Two forms of acetylcholinesterase, a membrane bound form as well as a soluble form, were found within these vesicles. The secretory vesicles, isolated by differential centrifugation, were further purified on a continuous isotonic Percoll? gradient. In this way, secretory vesicles were separated from mitochondrial, microsomal and cell membrane contamination. The secretory vesicles recovered from the gradient contained an average of 2.26 μmol adrenalin/mg protein. On incubation for 30 min at 37°C in media differing in ionic strength, pH, Mg²⁺ and Ca²⁺ concentration, the vesicles released less than 20% of total adrenalin. Acetylcholinesterase could hardly be detected in the secretory vesicle fraction when assayed in isotonic media. However, in hypotonic media (<400 mosmol/kg) or in Triton X-100 (0.2% final concentration) acetylcholinesterase activity was markedly higher. During hypotonic treatment or when secretory vesicles were specifically lyzed with 2 mM Mg²⁺ and 2 mM ATP, adrenalin as well as part of acetylcholinesterase was released from the vesicular content. On polyacrylamide gel electrophoresis this soluble enzyme exhibited the same electrophoretic mobility as the enzyme released into the perfusate from adrenal glands upon stimulation. In addition to the soluble enzyme a membrane bound form of acetylcholinesterase exists within secretory vesicles, which sediments with the secretory vesicle membranes and exhibits a different electrophoretic mobility compared to the soluble enzyme. It is concluded, that the soluble enzyme found within isolated secretory vesicles is secreted via exocytosis, whilst the membrane-bound form is transported to the cell membrane during this process, contributing to the biogenesis of the cell membrane.     

Subcellular localization and partial characterization of bovine corpus luteum adenylate cyclase.

The subcellular localization of adenylate cyclase (ATP pyrophosphatelyase (cyclizing), EC 4.6.1.1) in bovine corpus luteum was studied using isotonic and hypotonic homogenization and fractionation conditions. All fractions prepared were assayed for adenylate cyclase, marker enzymes and DNA. Only plasma membrane marker enzyme, 5'-nucleotidase paralleled the distribution of adenylate cyclase under both isotonic and hypotonic conditions (conditionsoth isotonic and hypotonic conditions (coefficient of correlation = 0.95). Two main fractions prepared under hypotonic conditions were subfractionated by discontinuous sucrose gradient centrifugation. The highest amount of adenylate cyclase was found in a fraction having a density approximately equal to 1.13 g/cm3. The specific activity of this fraction was 4--6 times higher than that of the homogenate. The electron microscopic study of this fraction revealed the presence of a single type of particulate material consisting of small vesicles exhibiting a typical unit membrane structure. It is concluded that this adenylate cyclase is primarily localized in the plasma membranes. Basal adenylate cyclase activity of plasma membranes was stimulated 2--3 times by luteinizing hormone (10 mug/ml), 3--4 times by prostaglandin E2 (10 mug/ml), 4--6 times by NaF (0.01 M) and two times by methanol (0.2%).     

The intracellular localization and properties of carnitine acetyltransferase from ram spermatozoa

Although spermatozoa possess a very active carnitine acetyltransferase, there is no satisfactory explanation for such a high activity. In order to help elucidate possible roles for carnitine acetyltransferase in spermatozoa, we examined the intracellular location and properties of carnitine acetyltransferase from ejaculated ram spermatozoa. The spermatozoa were disrupted by hypotonic treatment with 10 mm phosphate buffer (pH 7.4), followed by mild sonication. The resulting homogenate was separated by sucrose step-gradient centrifugation into soluble, plasma membrane, acrosomal membrane, and mitochondrial fractions. These fractions were characterized by electron microscopy and marker enzyme assays. The particulate fractions were made soluble by treatment with 0.1% deoxycholate and then were assayed for carnitine acetyltransferase activity. Carnitine acetyltransferase activity was found exclusively in the mitochondrial fraction with a specific activity of 0.151 μmol CoASH · min^?1 · mg^?1. The apparent K_m values for acetyl-CoA and l-carnitine were 1.1 × 10^?5 and 1.3 × 10^?4m respectively.     

Proliferating Cell Nuclear Antigen Bound to DNA Synthesis Sites: Phosphorylation and Association with Cyclin D1 and Cyclin A

Evidence is presented that association of proliferating cell nuclear antigen (PCNA) with nuclear chromatin in human fibroblasts is related to the phosphorylation status of the protein. Using a hypotonic lysis procedure to extract the soluble form of PCNA, it has been shown that the remaining nuclear-bound form, predominantly in S-phase cells, is highly phosphorylated. Cells in early G₁, or in G₂ + M phases, contain basal levels of the bound form of the protein that is only weakly phosphorylated. Using fractionated immunoprecipitation techniques, PCNA was found to be associated with cyclin A in both soluble and insoluble fractions. In contrast, association of PCNA with cyclin D1 was found in the soluble fraction, while no detectable levels were present in the insoluble fraction. Immunofluorescence labeling and flow cytometric analysis of the cell cycle distribution of cyclin D1 and cyclin A showed that, like PCNA, maximal levels of both proteins were bound to nuclear structures at the G₁/S phase boundary. These results suggest that binding of PCNA to DNA synthesis sites occurs after phosphorylation. Association with cyclin D1 and cyclin A might occur in a macromolecular complex assembled at the G₁/S phase boundary to drive activation of DNA replication factors.     

LOCAL CHANGES IN SUBCELLULAR DISTRIBUTION OF DOPAMINE-β-HYDROXYLASE (EC 1.14.2.1) AFTER BLOCKADE OF AXONAL TRANSPORT

Abstract— The distribution of DBH activity between soluble and sedimentable fractions of hypotonic homogenates was examined in rat sympathetic ganglia and nerves after interruption of axonal transport. Local application of colchicine to superior cervical ganglia caused an increase mainly in particulate DBH activity, which was presumably bound to membranes. Likewise, in sciatic nerves, particulate DBH activity accumulated on both sides of a ligature and disappeared from a region well below a ligature much faster than did soluble activity. On the other hand, 18 h after simultaneous application of two ligatures to the nerve, neither total DBH activity nor subcellular distribution of this activity changed in the isolated nerve region. More detailed analysis showed that ligation affected the distribution of DBH activity within a fraction that sedimented at 140,000 g after homogenization of nerves in isotonic sucrose. Just above a ligature, osmotically releasable DBH activity was a smaller proportion of the sedimentable activity than in control nerves. However, as compared to controls, osmotically releasable DBH activity was a larger proportion of the activity in the sedimentable fraction from a region well below a ligature. A model was developed which accounts for some of these results by postulating that DBH is associated with different compartments in sciatic nerve which have different rates of transport and different proportions of soluble and bound enzyme.     

ANTIGENS IN EGGS AND DEVELOPMENTAL STAGES OF THE SEA URCHIN : II. Localization

Homogenates of fertilized eggs of the sea urchin Paracentrotus lividus were fractionated by differential centrifugation. In addition, whole eggs were fragmented, on a preparative scale, by centrifugation in sea water-sucrose gradients. The fractions and fragments were subsequently assayed for their content of soluble protein antigens described in an earlier publication. Relative concentrations of antigen present in quantitatively isolated cell fractions were estimated by graded antiserum absorption in combination with agar-diffusion technique. Two of six antigens were found to be associated mainly with the low speed sediments. Treatment of the various sediments with hypotonic medium and results obtained with fragmented eggs suggested that these two antigens and possibly a third were probably located in the yolk granules. The other antigens were more evenly distributed among the low speed sediments and the non-sedimented part of the cytoplasm. Only one of the antigens was consistently associated with the microsomal fraction.     

Impact of hypotonic solutions on the stromal swelling, lactate dehydrogenase and aldehyde dehydrogenase activity of the keratocytes of the bovine cornea

The present studies were designed to explore the relationship between the swelling-related changes of the collagen-cell (keratocyte) matrix of the corneal stroma, and the integrity of the cells. From recent postmortem eyes of adult cattle, complete stroma preparations were dissected out and allowed to swell in solution (free swelling) or enclosed within a 12 kDa cut-off dialysis membrane with or without spacers. The swelling was at 4 degrees C with either water, a hypotonic phosphate-buffered saline (PBS, pH 7.0), a hypotonic mixed salt (MS) solution (pH 7.5), or an isotonic mixed salt solution with glucose (pH 7.5). Measures of tissue wet mass and thickness and analyses of the soluble protein, LDH and ALDH activity in the solutions were made. The relative swelling of the stroma preparations was greatest in water (to 624% of the original wet mass) > dilute PBS (to 404%) > dilute MS (to 381%) > MS with glucose (to 356%). The relative swelling was in the same order, but slightly less if the stroma preparations were enclosed in a dialysis tube with spacers, and substantially reduced when enclosed in a dialysis bag without spacers. With the use of hypotonic solutions, substantial quantities of proteinaceous material and enzyme activity were lost from the preparations, with the loss being proportional to the extent of swelling (p < 0.001). Swelling of an isolated corneal stroma, especially in hypotonic solutions, is associated with substantial loss of soluble protein and cytoplasmic enzyme activities, and so these solutions must be considered as cytotoxic to the keratocytes.     

Distribution of chloroquine in normal,pronase-treated and malaria-infected red cells

Pronase treatment of mouse red cells in the presence of chloroquine leads to greatly enhanced accumulation of the drug, which after freeze-thaw or hypotonic lysis is found to be located mainly in the membrane fraction. Much lower proportions of the drug are found in the membrane fraction prepared from Plasmodium berghei-infected red cells, which also have a high capacity for chloroquine accumulation. Pronase treatment of infected cells result only in a slight enhancement of total accumulation. The membrane-bound fraction of the drug is, however, increased while the fraction in the lysate is decreased. Membranes prepared from hypotonic lysis of normal or P. berghei-infected cells have similar capacities for chloroquine binding. These results show that the distribution of chloroquine in pronase-treated and malaria-infected cells are different and that pronase treatment of both normal and infected cells followed by lysis leads to availability of potential membrane binding sites.     

Nucleotide sequence of noncoding regions in Rous-associated virus-2: comparisons delineate conserved regions important in replication and oncogenesis.

Fujinami sarcoma virus (FSV) encodes a 140,000-dalton transforming protein, P140, which contains gag- and fps-specific sequences. The cellular localization of this protein was examined by fractionation of [35S]methionine-labeled, FSV-infected chicken embryo fibroblasts. In homogenates of cells infected by wild-type, temperature-resistant FSV prepared in either hypotonic or isotonic buffer, 60 to 80% of the P140 was particulate. Isopycnic separation on discontinuous sucrose gradients indicated that the majority of the particulate P140 was present in a light membrane fraction enriched for plasma membranes. Much of the particulate P140 could be solubilized by the addition of 0.6 M salt to a postnuclear supernatant, suggesting that P140 is not an integral membrane protein. Particulate P140 may be associated with membranes either directly as a peripheral membrane protein or indirectly via cytoskeletal elements. In cells infected by mutants of FSV temperature sensitive for cellular transformation, most of the P140 is particulate at the permissive temperature, whereas most is soluble at the nonpermissive temperature; this change in distribution is not a secondary consequence of the change in cellular phenotype, since it also occurs in nonconditionally transformed cells doubly infected with temperature-sensitive FSV and wild-type Rous sarcoma virus. The movement of P140 from the particulate to the soluble fraction occurs rapidly when cells infected by temperature-sensitive FSV are shifted from the permissive to the nonpermissive temperature. Furthermore, P140 moves from the soluble to the particulate fraction, although somewhat more slowly, when cells are shifted from the nonpermissive to the permissive temperature. These observations suggest that the association of P140 with plasma membranes or the cytoskeleton may play a role in transformation by FSV.     

T11: a new protein marker on activated murine T lymphocytes

Murine lymphocytes were activated in vitro in mixed lymphocyte cultures (MLC) or by the addition of the mitogens concanavalin A (Con A), phytohemagglutinin (PHA), or E. coli lipopolysaccharide (LPS). Activated lymphocytes were internally labeled with 35S-methionine and then disrupted by hypotonic lysis. A plasma membrane-enriched fraction was isolated from each cell population, and the 35S-labeled proteins in this fraction were examined by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). An intensely labeled band, the position of which indicated an apparent m.w. of 11,000, was observed when plasma membrane-enriched fractions from MLC- and Con A-activated cells were subjected to SDS-PAGE. In contrast, plasma membrane-enriched fractions from normal spleen cells, LPS-activated cells, PHA-activated cells, and EL4, RDM4, and P815 tumor cells possessed little or none of this protein, which we have designated T11. T11 was not found in the soluble cytoplasmic protein from MLC-activated cells. Hence the presence of T11 in the plasma membrane-enriched fraction from these cells cannot be attributed to contamination by cytoplasmic protein. Removal of T cells from populations of MLC-activated cells by treatment with monoclonal anti-Thy 1 and complement removed T11. These results suggest that T11 may represent a new protein marker on a subclass of activated T lymphocytes.     

Multiple forms of cyclic nucleotide phosphodiesterase in pig epidermis.

Pig epidermal cyclic nucleotide phosphodiesterases (EC 3.1.4.16) have been partially purified by DEAE-cellulose column chromatography. At least three different forms of the epidermal phosphodiesterases were identified. They were cyclic GMP-specific, cyclic GMP- and cyclic AMP-hydrolyzing and apparently a cyclic AMP-specific enzyme: the first two forms were soluble and the last was the particulate enzyme. The cyclic GMP-specific soluble fraction had a relatively low Km, the cyclic GMP- and cyclic AMP-hydrolyzing fraction had a high Km for the respective substrates and the third particulate enzyme had both high and low Km values for cyclic AMP. The cyclic GMP-hydrolyzing enzyme was localized almost entirely in the soluble fraction, whereas cyclic AMP-hydrolyzing enzyme was distributed to both soluble and particulate fractions. Thus, our studies show that the multiple forms of pig epidermal enzyme differ distinctly in their substrate affinity, specificity and subcellular distribution.     

Binding of nascent collagen by amyloidogenic light chains and amyloid fibrillogenesis in monolayers of human fibrocytes

Light (L) chain dimers expressed by multiple myeloma cells and collected as Bence-Jones proteins from the urine of human subjects were tested for their ability to form deposits in fibroblast monolayer cell cultures. Bence-Jones proteins from subjects with primary amyloidosis associated with L chains were shown to form fibrillar deposits by the in vitro assay introduced in this report. Filaments interspersed with nascent collagen could be detected after only 48 h. Deposition of L chains continued over a period of 72 h culminating in the appearance of dense fibrils with widths of 80-100 A and a variety of lengths. Formation of amyloid-like fibrils was accompanied by interference with the maturation of the collagen produced by the fibroblast cells. Fibrils composed of the Mcg lambda-type L chain were deposited between collagen fibers, thus expanding them laterally and leading to their partial disintegration. Mature collagen was completely missing from fibroblast monolayers exposed to the Sea lambda chain and the Jen kappa chain. Collagen with the characteristic striped pattern matured normally in control samples, such as those not dosed with amyloid precursors or those treated with a non-amyloidogenic Bence-Jones protein (e.g., the Hud lambda chain dimer). By immunochemical techniques using fluorescein- and gold-labeled anti-L chain antibodies, amyloidogenic L chains were shown to decorate the strands of nascent collagen. This observation suggests that amyloidogenic L chains are concentrated in the extracellular matrix by monovalent antigen-antibody type reactions. The capacity of the Mcg L chain dimer to bind collagen-derived sequences was tested by soaking crystals with a collagenase substrate, PZ-Pro-Leu-Gly-Pro-D-Arg. Difference Fourier analysis at 2.7 A resolution indicated that the PZ-peptide is a site-filling ligand. It could not be removed from the active site by perfusion of the crystal with ammonium sulfate crystallizing media. Similar experiments with the collagen-derived peptide (Pro-Pro-Gly)(5) showed substantial hysteresis effects extending from one end of the Mcg dimer to the other. After the ligand was withdrawn, the active site of the Mcg dimer could no longer bind the PZ-peptide. However, if the active site was first blocked by the PZ-peptide and subsequently exposed to the (Pro-Pro-Gly)(5) peptide, the difference Fourier map was indistinguishable from that obtained with the PZ-peptide alone. We concluded that amyloidogenic L chains such as the Mcg dimer could be concentrated in the perivascular space by binding to normal tissue constituents. These components include nascent collagen, which can be deterred from maturing as a result of this binding. Participation in such pathological activity is also self-destructive to the amyloidogenic L chains, which lose their binding capabilities for collagen-derived peptides and also become susceptible to irreversible conversion to amyloid fibrils. All of these events may be prevented by prior treatment of the amyloidogenic L chains with site-filling ligands. (c) 2000 John Wiley & Sons, Ltd.     

Metabolism of the reserve polysaccharide of Streptococcus mitior (mitis): is there a second alpha-1,4-glucan phosphorylase?

The alpha-1,4-glucan phosphorylase (alpha-1,4-glucan: orthophosphate glucosyltransferase; EC 2.4.1.1) associated with the particulate cell fraction of Streptococcus mitior strain S3 was compared with the soluble maltodextrin phosphorylase that had been previously isolated from the same organism (Walker et al., 1969). The particulate enzyme was more sensitive to the glycogen content of the cell than the soluble euzyme; its activity was highest when the cells were grown under conditions favoring high glycogen storage. Substrate specificities of the two high activity towards endogenous glycogen, whereas low-molecular-weight maltodextrins were the preferred substrates for the soluble phosphorylase. The purification of the particulate phosphorylase included incubation of the particulate fraction in 160 mM sodium phosphate-10 mM sodium citrate-0.1% (wt/vol) Triton X-100 buffer (pH 6.7) and ion-exchange chromatography on diethylamino-ethyl- Sephadex A-50. The purified enzyme was fully soluble. The value for the purification factor was variable and depended on (i) the substrate used and (ii) whether the synthetic or the degradative reaction was being measured. The solubilization resulted in considerable changes in the properties of the phosphorylase: the pH optimum for activity was raised from 6.0 to 7.0-7.5 and the substrate specificity was altered. Consequently, the purified enzyme bore greater similarity to the soluble maltodextrin phosphorylase. The reported results are best explained in terms of a single phosphorylase, the specificity which is determind by its binding state in the cell. The enzyme acts as a glycogen phosphorylase in the particulate state and as a maltodextrin phosphorylase when soluble. The equilibrium between the two forms is related to the glycogen content of the cells.     

Activation of guanylate cyclase in cerebral cortex of rat by hydroxylamine.

Hydroxylamine actived guanylate cyclase in particulate fraction of cerebral cortex of rat. Activation was most remarkable in crude mitochondrial fraction. When the crude mitochondrial fraction was subjected to osmotic shock and fractionated, guanylate cyclase activity recovered in the subfractions as assayed with hydroxylamine was only one-third of the starting material. Recombination of the soluble and the particulate fractions, however, restored guanylate cyclase activity to the same level as that of the starting material. When varying quantities of the particulate and soluble fractions were combined, enzyme activity was proportional to the quantity of the soluble fraction. Heating of the soluble or particulate fraction at 55 degrees for 5 min inactivated guanylate cyclase. The heated particulate fraction markedly activated guanylate cyclase activity in the native soluble fraction, while the heated soluble fraction did not stimulate enzyme activity in the particulate. The particulate fraction preincubated with hydroxylamine at 37 degrees for 5 min followed by washing activated guanylate cyclase activity in the soluble fraction in the absence of hydroxylamine. Further fractionation of the crude mitochondrial fraction revealed that the factor(s) needed for the activation by hydroxylamine is associated with the mitochondria. The mitochondrial fraction of cerebral cortex activated guanylate cyclase in supernatant of brain, liver, or kidney in the presence of hydroxylamine. The mitochondrial fraction prepared from liver or kidney, in turn, activated soluble guanylate cyclase in brain. Activation of guanylate cyclase by hydroxylamine was compared with that of sodium azide. Azide activated guanylate cyclase in the synaptosomal soluble fraction, while hydroxylamine inhibited it. The particulate fraction preincubated with azide followed by washing did not stimulate guanylate cyclase activity in the absence of azide. The activation of guanylate cyclase by hydroxylamine is not due to a change in the concentration of the substrate GTP, Addition of hydroxylamine did not alter the apparent Km value of guanylate cyclase for GTP. Guanylate cyclase became less dependent on manganese in the presence of hydroxylamine. Thus the activation of guanylate cyclase by hydroxylamine is due to the change in the Vmax of the reaction.