Association of beta-glucosidase with intact cells of Thermoactinomyces.

The location of the B-glucosidase activity in a whole culture broth of the thermophilic organism Thermoactinomyces has been studied. Little beta-glucosidase activity was found in the culture filtrate, while the culture solids contained the major part of the activity of the whole culture broth. The activity does not appear to be adsorbed to the culture solids; rather there is evidence that it is an intracellular soluble enzyme(s). The pH and temperature optima for a crude beta-glucosidase preparation were determined to be pH 6.5 and 50--55 degrees C. Enzyme activity studies indicate that the same enzyme(s) accounts for the beta-glucosidase and the cellobiase activities. The validity of using the filter paper activity of culture filtrates from Thermoactinomyces to predict the total saccharification of cellulosic materials to glucose is discussed.     

Gene expression in Brassica campestris showing a hypersensitive response to the incompatible pathogen Xanthomonas campestris pv. vitians

Xanthomonas campestris pv. vitians, a pathogen of lettuce, elicits a hypersensitive response within 12 hours of inoculation into Brassica leaves, characterized by tissue collapse, loss of membrane integrity, vein blockage and melanin production. In contrast, the compatible pathogen, X. c. pv. campestris, has no visible effects on leaves for 48 hours, after which inoculated areas show chlorosis which eventually spreads, followed by rotting.mRNA was prepared from leaves inoculated with suspensions of both pathovars or with sterile medium up to 24 hours following inoculation. In vitro translation of total and poly A⁺ RNA in rabbit reticulocyte lysate in the presence of ³⁵S methionine followed by separation of the polypeptide products by 2D-PAGE, allowed comparison of the effects of these treatments on plant gene expression. Major changes in gene expression were observed as a consequence of the inoculation technique. In addition, after inoculation with X. c. vitians, up to fifteen additional major polypeptides appeared or greatly increased by four hours. Some of these had disappeared by nine hours and several more had appeared. No major polypeptides disappeared or decreased greatly in intensity following inoculation with X. c. vitians.     

Uterine blood flow during the development and regression of the decidual cell reaction in ovariectomized, steroid-treated mice

Uterine blood flow was assessed in mice by measuring organ uptake of intravenously injected [14C]butanol. In ovariectomized mice, injection of 100 ng oestradiol-17 beta increased blood flow 5-fold over that of untreated controls. The injection of oestradiol-17 beta in progesterone-treated mice also increased uterine blood flow at the time of maximal sensitivity to a decidual stimulus, but not 4 days later. Absolute values of blood flow increased during development of the decidual cell reaction in proportion to the increase in uterine weight, reaching maximal values 96 h after decidual induction. When progesterone injections were stopped 72 h after decidual induction, a rapid decrease in absolute and relative blood flow values preceded the decrease in uterine weight. This decrease in uterine blood flow occurred within 12 h of removing a subcutaneous implant containing progesterone. These results are consistent with the view that increased uterine blood flow during decidual development may be necessary to support the rapid increase in uterine weight at implantation and the subsequent decrease in both relative and absolute uterine blood flow on withdrawal of progesterone may promote decidual regression in the mouse.     

Structural relationships of actin, myosin, and tropomyosin revealed by cryo-electron microscopy

We have calculated three-dimensional maps from images of myosin subfragment-1 (S1)-decorated thin filaments and S1-decorated actin filaments preserved in frozen solution. By averaging many data sets we obtained highly reproducible maps that can be interpreted simply to provide a model for the native structure of decorated filaments. From our results we have made the following conclusions. The bulk of the actin monomer is approximately 65 X 40 X 40 A and is composed of two domains. In the filaments the monomers are strongly connected along the genetic helix with weaker connections following the long pitch helix. The long axis of the monomer lies roughly perpendicular to the filament axis. The myosin head (S1) approaches the actin filament tangentially and binds to a single actin, the major interaction being with the outermost domain of actin. In the map the longest chord of S1 is approximately 130 A. The region of S1 closest to actin is of high density, whereas the part furthest away is poorly defined and may be disordered. By comparing maps from decorated thin filaments with those from decorated actin, we demonstrate that tropomyosin is bound to the inner domain of actin just in front of the myosin binding site at a radius of approximately 40 A. A small change in the azimuthal position of tropomyosin, as has been suggested by others to occur during Ca2+-mediated regulation in vertebrate striated muscle, appears to be insufficient to eclipse totally the major site of interaction between actin and myosin.     

Tissue-specific regulation of GTP-binding protein and muscarinic acetylcholine receptor levels during cardiac development

A quantitative immunoblot assay was developed by using affinity-purified monospecific antibodies to quantitate levels of guanine nucleotide binding regulatory protein (G-protein) subunits in atria and ventricles during embryonic chicken cardiac development. The muscarinic acetylcholine receptor (mAChR) number was measured with [3H]quinuclidinyl benzilate. On day 10 of embryonic development (day 10E) there was no difference between the atrial and ventricular membrane concentrations of beta-subunit, G0 alpha subunit, or mAChR. The level of Gi alpha was found to be 44% greater in atria than in ventricles on day 10E. The atrial membrane concentration of beta-subunit increased 80% between day 13E and 15E, G0 alpha increased 46% between day 10E and 15E, mAChR increased 61% between day 10E and 12E, and Gi alpha decreased 34% between day 10E and 13E. The atrial levels of beta-subunit, G0 alpha, Gi alpha, and mAChR did not change further through day 20E. The ventricular membrane concentration of these proteins did not change between day 10E and 20E, except for that of G0 alpha, which increased 47% between day 15E and 20E. The atrial specific increase in beta-subunit correlated with a loss of GTP inhibition of basal adenylate cyclase activity. The difference in Gi alpha levels between atria and ventricles on day 10E correlated with a difference in carbachol sensitivity of atrial and ventricular basal adenylate cyclase activity. Thus, the levels of several components of the cholinergic neuroeffector pathway are regulated in a tissue-specific manner at a time that coincides with the onset of functional parasympathetic innervation of the embryonic chicken heart.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chemically induced hypothyroidism produces elevated amounts of the alpha subunit of the inhibitory guanine nucleotide binding protein (Gi) and the beta subunit common to all G-proteins.

Adipocytes of hypothyroid rats display an increased responsiveness to agents which function by inhibiting the production of cyclic AMP. Anti-peptide antisera which selectively recognise the alpha subunit of the inhibitory guanine nucleotide binding protein (Gi) detected a 40 kDa polypeptide in adipocyte plasma membranes of both euthyroid and hypothyroid rats. Amounts of the alpha subunit of Gi were elevated some 2-fold in the hypothyroid preparations in comparison with the euthyroid controls, when equal amounts of membrane protein of the two treatments were examined. As cells from the hypothyroid animals contained 2.7 times as much membrane protein as those from the control animals, the amounts of alpha subunit of Gi are elevated some 5.6-fold per cell in adipocytes of the hypothyroid animals compared with the euthyroid controls. Amounts of the 36 kDa beta subunit of G-proteins were also elevated in plasma membranes of adipocytes of hypothyroid animals, in this case by some 50% when compared on a protein basis. These results provide direct evidence for alterations in the amounts of the subunits of Gi caused by the hypothyroid state.     

Adipocyte plasma-membrane Gi and Gs in insulinopenic diabetic patients.

The matrix method for calculating the overall sensitivities (including control coefficients) of a metabolic system, described by Crabtree & Newsholme [Biochem. J. 247, 113-129 (1987)], is simplified by a preliminary partitioning of the initial matrix equation. This reduces the size of the matrix to be inverted and thereby removes a major drawback with the original method. The resulting procedure is simpler and more systematic than the alternative methods currently available, especially when the system is extensively branched.     

Glucagon desensitization of adenylate cyclase and stimulation of inositol phospholipid metabolism does not involve the inhibitory guanine nucleotide regulatory protein Gi, which is inactivated upon challenge of hepatocytes with glucagon.

Brief exposure of hepatocytes to glucagon, angiotensin or the protein kinase C activator TPA (12-O-tetradecanoylphorbol 13-acetate) caused the inactivation of the inhibitory guanine nucleotide regulatory protein Gi. Glucagon-mediated desensitization of glucagon-stimulated adenylate cyclase activity was seen in hepatocytes from both normal rats and those made diabetic with streptozotocin, where Gi is not functionally expressed. Normal glucagon desensitization was seen in hepatocytes from young animals, 6 weeks of age, which had amounts of Gi in their hepatocyte membranes which were some 45% of that seen in mature animals (3.4 pmol/mg of plasma-membrane protein). Streptozotocin-induced diabetes in young animals abolished the appearance of functional Gi in hepatocyte plasma membranes. Pertussis-toxin treatment of hepatocytes from both normal mature animals and those made diabetic, with streptozotocin, blocked the ability of glucagon or angiotensin or TPA to elicit desensitization of adenylate cyclase. The isolated B (binding)-subunit of pertussis toxin was ineffective in blocking desensitization. Neither induction of diabetes nor treatment of hepatocytes with pertussis toxin inhibited the ability of glucagon and angiotensin to stimulate the production of inositol phosphates in intact hepatocytes. Thus (i) Gi does not appear to play a role in the molecular mechanism of glucagon desensitization in hepatocytes, (ii) the G-protein concerned with receptor-stimulated inositol phospholipid metabolism in hepatocytes appears not to be a substrate for the action of pertussis toxin, (iii) in intact hepatocytes, treatment with glucagon and/or angiotensin can elicit the inactivation of the inhibitory G-protein Gi, and (iv) pertussis toxin blocks desensitization by a process which does not involve Gi.     

Persistent activation of the alpha subunit of Gs promotes its removal from the plasma membrane.

As assessed both by cholera-toxin-catalysed ADP-ribosylation and by immunoblotting with an anti-peptide antiserum raised against the C-terminal decapeptide of forms of Gs alpha (the alpha subunit of the stimulatory guanine nucleotide-binding protein), rat glioma C6 BU1 cells express two forms of Gs alpha: a major 44 kDa form and a much less prevalent 42 kDa form. We examined the effects of guanine nucleotides on the interaction of the 44 kDa form with the plasma membrane. Incubation of membranes of C6 BU1 cells with poorly hydrolysed analogues of GTP, but not with analogues of either ATP or GDP, caused the release of this Gs alpha from the membrane fraction. Release of Gs alpha was observed within 5 min, and continued throughout the incubation period. After treatment with guanosine 5'-[beta gamma-imido]triphosphate for 60 min, some 75% of this polypeptide had been released from its site of membrane attachment. These experiments demonstrate that Gs alpha need not remain associated invariantly with the plasma membrane.     

Cholera toxin treatment produces down-regulation of the alpha-subunit of the stimulatory guanine-nucleotide-binding protein (Gs).

The effect of activation of the alpha-subunit(s) of the stimulatory guanine-nucleotide-binding protein, Gs, on levels of this polypeptide(s) associated with the plasma membrane of L6 skeletal myoblasts was ascertained. Incubation of these cells with cholera toxin led to a time- and concentration-dependent 'down-regulation' of both 44 and 42 kDa forms of Gs alpha as assessed by immunoblotting with an anti-peptide antiserum (CS1) able to identify the extreme C-terminus of Gs. The effect of cholera toxin was specific for Gs; levels of Gi alpha in membranes of cholera toxin-treated cells were not different from untreated cells. Down-regulation of Gs was absolutely dependent upon prior ADP-ribosylation, and hence activation of Gs and was not mimicked by other agents which elevate intracellular levels of cyclic AMP. Pretreatment with pertussis toxin, which catalyses ADP-ribosylation of Gi but not of Gs, did not down-regulate either Gi or Gs, demonstrating that covalent modification by ADP-ribosylation is alone not a signal for removal of G-proteins from the plasma membrane.