Glenn D. Kuehn

Polyacrylamide-boronate beads saturated with biomolecules: A new general support for affinity chromatography of enzymes

Aminoethyl polyacrylamide beads (P-150) were quantitatively coupled to m-aminobenzeneboronic acid to yield a product containing 1.3 mmoles of boronic acid per gram (dry weight) of gel. The average pKa of the insolubilized boronic acid was determined to be 9.2. At pH 8.45 a number of enzyme substrates and cofactors, including NAD+, citric acid, pyridoxal, and epinephrine, were shown to bind to P-150-boronate beads packed in chromatography columns. At saturation, the P-150-boronate beads bound 2.5 to 80 μmoles of substrate or cofactor per ml of wet packed gel. A P-150-boronate column saturated with uridine 5′-triphosphate was used to achieve a 1000-fold purification of the enzyme uridine 5′-diphosphate glucose pyrophosphorylase (EC 2.7.7.9) from the slime mold, Physarum polycephalum.

Putrescine Aminopropyltransferase Is Responsible for Biosynthesis of Spermidine, Spermine, and Multiple Uncommon Polyamines in Osmotic Stress-Tolerant Alfalfa

The biosynthesis of polyamines from the diamine putrescine is not fully understood in higher plants. A putrescine aminopropyltransferase (PAPT) enzyme activity was characterized in alfalfa (Medicago sativa L.). This enzyme activity was highly specific for putrescine as the initial substrate and did not recognize another common diamine, 1,3-diaminopropane, or higher-molecular-weight polyamines such as spermidine and spermine as alternative initial substrates. The enzyme activity was inhibited by a general inhibitor of aminopropyltransferases, 5[prime]-methylthioadenosine, and by a specific inhibitor of PAPTs, cyclohexylammonium sulfate. The initial substrate specificity and inhibition characteristics of the enzyme activity suggested that it is a classical example of a PAPT. However, this enzyme activity yielded multiple polyamine products, which is uncharacteristic of PAPTs. The major reaction product of PAPT activity in alfalfa was spermidine. The next most abundant products of the enzyme reaction using putrescine as the initial substrate included the tetramines spermine and thermospermine. These two tetramines were distinguished by thin-layer chromatography to be distinct reaction products exhibiting differential rates of formation. In addition, the uncommon polyamines homocaldopentamine and homocaldohexamine were tentatively identified as minor enzymatic reaction products but only in extracts prepared from osmotic stresstolerant alfalfa cultivars. PAPT activity from alfalfa was highest in meristematic shoot tip and floral bud tissues and was not detected in older, nonmeristematic tissues. Product inhibition of the enzyme activity was observed after spermidine was added into the in vitro assay for alfalfa PAPT activity. A biosynthetic pathway is proposed that accounts for the characteristics of this PAPT activity and accommodates a novel scheme by which certain uncommon polyamines are produced in plants.

Assay of adenylate cyclase by use of polyacrylamide--boronate gel columns.

A method is described for covalent attachment of up to 1.1 mmol of m-aminobenzeneboronic acid/g dry weight of polyacrylamide beads. Small (4 ml) columns of the derivatized beads have been used to separate quantitatively cyclic AMP from ATP in a single-step procedure. Columns of the polyacrylamide-boronate gel have been used as the basis of a convenient new assay of the soluble adenylate cyclase prepared from nuclei of Physarum polycephalum.

INHIBITION OF ORNITHINE DECARBOXYLASE IN HUMAN FIBROBLAST CELLS BY TYPE I AND TYPE II INTERFERONS

Dilution of human fibroblast GM2767 cell cultures into fresh serum-containing growth medium induces ornithine decarboxylase activity 45-fold over a six-hour interval. When the fibroblast cultures are supplemented with human fibroblast alpha-, beta-, or gamma-interferon at the time of dilution into fresh growth medium, the induction of ornithine decarboxylase is inhibited 61%, 90%, and 65%, respectively. beta-Interferon is the most effective type of interferon to inhibit induction of ornithine decarboxylase.

Role of Polyamines in Apoptosis and Other Recent Advances in Plant Polyamines

Numerous citations in the literature indicate that polyamines are intensively studied in plants. Polyamines are implicated in many functions of the plant cell. Excellent recent reviews cover much of this original research. This article is focused primarily on literature that relates research on the role of polyamines in apoptosis and programmed cell death in both plants and animals. Apoptosis and programmed cell death are considerably better studied in animal systems, and this review demonstrates that the role of polyamines in these processes in plant systems are remarkably congruent with what is known in animal systems. In addition, key recent research reports are reviewed that describe the functional analysis of key polyamine biosynthesis genes in plants in relation to responses to environmental stress signals. Molecular analysis is providing strong evidence for the polyamine biosynthetic pathways to play major roles in ameliorating plant responses to abiotic stresses.

Evidence for the occurrence of polyamine oxidase in the dicotyledonous plant Medicago sativa L. (alfalfa).

Polyamine oxidase (EC 1.5.3.3) activity has not been detected previously in cells of dicotyledonous plants, although it has been characterized extensively in monocotyledonous plants. Evidence is presented in this report for the occurrence of polyamine oxidase in dialyzed crude extracts of the dicotyledonous plant, Medicago sativa L. (alfalfa). Three enzyme assays were used to quantitate the formation of the three products of the reaction catalyzed by polyamine oxidase. 1-Pyrroline formation was measured colorimetrically as a yellow quinazolinium complex with o-aminobenzaldehyde. Hydrogen peroxide formation was measured spectrophotometrically with a coupled peroxidase assay system by peroxidative oxidation of guaiacol. [3H]1,3-Diaminopropane formation was measured by using [1,8-3H]spermidine as the substrate and separating the radiolabelled reaction product from the substrate by paper electrophoresis. This latter assay provided evidence that a polyamine oxidase of type [EC 1.5.3.3] catalyzed the cleavage reaction between a secondary nitrogen atom and an adjacent carbon of the butyl moiety of spermidine. Significant polyamine oxidase activity was detected in floral tissues, cortex tissues of the root, young leaves, and young germinated seedlings of alfalfa. The occurrence of polyamine oxidase in alfalfa accounts for the formation of the essential substrate, 1,3-diaminopropane, required for the biosynthesis of the uncommon polyamines, norspermidine and norspermine, which we have recently detected in alfalfa.

Genetics, subcellular localization, and molecular characterization of 6-phosphogluconate dehydrogenase isozymes in tomato.

Three independent genes are responsible for 6-phosphogluconate dehydrogenase (6PGDH) activity in tomato. 6Pgdh-2, located on chromosome 12, codes for subunits of an active dimer which is restricted to the plastids. 6Pgdh-1, chromosome 4, and 6Pgdh-3, chromosome 5, code for subunits which form three dimers—two homodimers and an intergenic heterodimer. The latter three isozymes are found in the cytosol. 6Pgdh-1, 6Pgdh-2, and 6Pgdh-3 code for subunits with estimated molecular weights of 49,500, 50,500, and 51,200, respectively. The intergenic heterodimer encoded by 6Pgdh-1 and 6Pgdh-3 is thus composed of subunits that differ in length by approximately 15 amino acid residues. Divergence in the length and primary subunit structure may account for the lower thermal stability of the intergenic heterodimer compared with the corresponding homodimers. A limited survey of other solanaceous plant species suggests that the duplication of cytosolic 6PGDH-coding genes found in tomato may be widespread in the family.

Cell cycle variation in cyclic adenosine 3′, 5′-monophosphate-dependent inhibition of a protein kinase from Physarum,polycephalum

Abstract A protein kinase present in the acellular slime mold, Physarum , polycephalum , has been found to exhibit cell cycle dependence with respect to inhibition by cyclic adenosine 3′,5′-monophosphate (cyclic AMP). The capacity of 1 × 10−5 M cyclic AMP to inhibit the kinase was maximal (∼80%) during G2 phase. At the onset of mitosis, a sharp rate of decrease occurred in the inhibitory response to cyclic AMP which continued for approximately one hour to mid-S phase. For approximately one hour during mid-S phase, the kinase activity was independent of cyclic AMP concentration. There-after, the capacity of cyclic AMP to inhibit the protein kinase was restored over a two-hour interval. Complete restoration coincided with the termination of S phase. Protein kinase levels measured in these same preparations in the absence of cyclic AMP remained invariant throughout the cell cycle. Thus, control of the protein kinase is apparently achieved through coordinate action of cyclic AMP plus other un-identified factors rather than by differential synthesis and destruction of a single kinase enzyme.

Interferon induction of polyamine-dependent protein kinase activity in ehrlich ascites tumor cells.

Abstract Treatment of Ehrlich ascites tumor cell cultures in vitro with interferon induces a protein kinase activity that is activated by the polyamines, spermidine and spermine. Putrescine antagonizes the activation. The protein kinase yields a phosphorylated endogenous polypeptide of M r 68,000–70,000. The polyamine-dependent protein kinase activity cofractionates with a double-stranded RNA-dependent protein kinase activity during affinity chromatography on poly (I) ·poly (C) - agarose or by chromatography on phosphocellulose. The double-stranded RNA-dependent protein kinase also phosphorylates an endogenous polypeptide of M r 68,000–70,000. Unsuccessful attempts to discriminate between these two protein kinase activities on the bases of their respective capacities to be activated by either double-stranded RNA or spermidine/spermine, suggest that a single protein kinase enzyme may be activated by these strikingly dissimilar modifiers.

Adenylate cyclases in physarum polycephalum: inhibition of a nuclear enzyme by polyamines.

Abstract Two distinct adenylate cyclase enzymes have been found in Physarum polycephalum . One is derived from isolated nuclei and is potently inhibited by an equimolar combination of the three polyamines, putrescine, spermidine, and spermine. The second enzyme is particulate, derived from the cytoplasmic compartment, and is insensitive to inhibition by the polyamines. These observations support a potential role for the polyamines in the control of adenosine 3′,5′-monophosphate synthesis in P. polycephalum nuclei.