Herb B. LéJohn

Cytokinins regulate calcium binding to a glycoprotein from fungal cells

Abstract A glycoprotein that binds about 20 atoms of Ca per mole has been purified from the osmotic shock fluid of some unicellular coenocytic water-molds, Achlya spp. and Blastocladiella emersonii . The binding of calcium is allosterically regulated by N6-(substituted)adenine derivatives, cytokinins. Pyrimidines, purine and pyrimidine nucleosides, auxins, and benzimidazole derivatives are ineffective in inhibiting calcium binding. Lysozyme partially inactivates the molecule so that a high affinity calcium binding site is destroyed. Trypsin and pronase inactivate the molecule so that Ca++ binding to both high and low affinity sites is affected. Cytokinins inhibit calcium binding to both sites.

Phosphorylated guanosine derivatives of eukaryotes: Regulation of DNA-dependent RNA polymerases I, II, and III in fungal development

Abstract Three phosphorylated guanosine derivatives designated HS-1, HS-2 and HS-3 synthesised during active protein synthesis in the water-mould, Achlya sp (1969) were shown to regulate the enzymatic activities of nucleoplasmic and nucleolar DNA-dependent RNA polymerases (RNAP-I, II and III) from both Achlya and another unrelated water-mould, Blastocladiella emersonii . These HS compounds were without effect on E. coli DNA-dependent RNA polymerase holoenzyme. The most potent of the three compounds was HS-3 which inhibited the activity of all enzymes completely at 100 μg/ml. HS-1, on the other hand, activated maximally at 1 to 10 μg/ml. HS-1 activation (3-fold) was restricted to enzyme III, and it had only partial inhibitory effects on enzymes I and II. The pattern of synthesis of HS-compounds throughout the 20-hour asexual growth cycle of the organism correlated with the detectable levels of the different RNA polymerases of Achlya .

Regulation of fungal ribonucleotide reductase by unusual dinucleotides.

Abstract Three phosphorylated dinucleosides designated HS1, HS2, and HS3, isolated from the water-mould Achlya , were shown to significantly inhibit ribonucleotide reductase activity from Achlya . All three compounds decreased CDP reduction in fungal extracts by 50% at concentrations of 0.1mM. At the same concentration HS3 also inhibited partially purified CDP reductase from Chinese hamster ovary cells by at least 80% but showed only 10% inhibition with enzyme from E. coli . ADP reductase activity from Achlya was inhibited 50% by both HS1 and HS3 at 0.1mM. HS2 however, showed no inhibitory effect on purine reduction. The levels of ribonucleotide reductase during the asexual growth cycle of Achlya correlated with thymidine uptake into DNA and with the synthesis of HS compounds.

Diguanosine nucleotides of fungi that regulate RNA polymerases isolated and partially characterised

Three unusual phosphorylated diguanosine compounds called ‘hot spots’ HS-1, HS-2 and HS-3 (ref. LeJohn, H.B. Proc. Can. Fed. Biol. Soc. 18, 159, 1975) have been isolated as acid-soluble materials from several fungi, Achlya, Blastocladiella emersonii, Aspergillus niger and Rhizopus stolonifer in their vegetative phase. The nucleotides were purified from acid extracts of Achlya and Blastocladiella. The tentative structures of HS-3 and HS-2 determined are GppppG and GppppGp. HS-1 structure is still in doubt but it is related to HS-2. The structures were deduced from enzymatic digestion and UV analyses of the products, molar ratios of guanosine and phosphate, and chromatographic behaviour on PEI-cellulose. All three compounds accumulated in an inverse manner with rates of RNA synthesis and directly with rates of protein synthesis. The acid-soluble pools of the three compounds fluctuated during the life cycle of Achlya, and just prior to sporulation, were excreted into the medium. HS-2 was convertible to HS-3 by acid hydrolysis.

Genetical and biochemical evidence that a novel dinucleoside polyphosphate coordinates salvage and de novo nucleotide biosynthetic pathways in mammalian cells.

Abstract Studies with ‘wild type’ Chinese hamster ovary cells and mutant derivatives defective in purine salvage and de novo nucleotide biosynthesis pathways have brought to light the possibility that an unusual dinucleoside polyphosphate, HS-3 (see appendix) is a crucial regulator of these two pathways. Three antitumor drugs, methotrexate, 5-fluorouracil and azaserine as well as L-glutamine, purines and pyrimidines were used to define the loci of HS-3 metabolism. Wild type and salvage pathways mutants accumulated HS-3 in the absence of glutamine. De novo pathways mutant accumulated HS-3 only when purine was absent. Depletion of HS-3 was induced in wild type and de novo mutant cell lines by purine compounds. Salvage pathways mutants did not cause depletion of HS-3 when supplied with purines or pyrimidines, except 5-fluorouracil. Data indicate that HS-3 is probably synthesised when an early step in purine nucleotide synthesis is blocked and depleted when the salvage pathways are operative. HS-3 may be an important factor in certain diseases involving nucleotide metabolism.

Relationship between uridine nucleotide sugar activation of glutamic dehydrogenases in fungi and existence of chitin and cellulose in their walls

Abstract Uridylates, uridine nucleotide sugars and uridine nucleotide amino sugars function as allosteric activators of DPN-linked glutamic dehydrogenases of some fungi. The effect appears to be restricted to glutamic dehydrogenases obtained from those ‘cellulosic’ fungi that do not synthesize chitin in their cell walls. These glutamic dehydrogenases have also retained the ability to interact with five other activators that had been found for all members of the Oomycetes (LeJohn, Stevenson and Meuser, J. Biol. Chem. 245 . 5569, 1970).

Involvement of protease in L-glutamine control of nucleic acid and polyphosphate metabolism in cells transformed by 9,10-dimethyl-1,2-benzanthracene, SV40 and H-ras oncogene

Mammalian cells transformed with either 9,10-dimethyl-1,2-benzanthracene, SV40 or H-ras oncogene dramatically changed their ability to synthesize DNA and RNA and metabolize polyphosphate when L-glutamine was withdrawn from the growth medium or when heat shocked (growth at 42 degrees C). Untransformed, DNA and RNA synthesis decreased by 50-80% when glutamine was withdrawn, but polyphosphate accumulated whether or not glutamine was supplied. Heat shock did not alter this response. Transformed isogenic cells responded differently; at 37 degrees C, they decreased their synthesis of DNA and RNA if starved for glutamine, whereas at 42 degrees C, synthesis was optimal without glutamine. Transformed cells accumulated polyphosphate at 37 degrees C when starved for glutamine, but at 42 degrees C, no polyphosphate accumulated. This apparent non-dependence on glutamine by transformed cells when heat shocked was found to be due to the production of glutamine from serum proteins through induction of a protease(s).

HS3 - A BIZARRE DINUCLEOSIDE POLYPHOSPHATE AS POSSIBLE PLEIOTYPIC REGULATOR OF EUKARYUTES

A bizarre dinucleoside polyphosphate, HS3, accumulated rapidly in fungal and established mammalian (permanent) cells in culture when they were starved of L-glutamine. Restoration of glutamine caused HS3 to decay in minutes. The effect was specific for glutamine among all protein amino acids. This effect of glutamine was antagonised by P i and, it then acted as a stimulant for HS3 synthesis. When glutamine induced HS3 decay in the fungal cell, sporogenesis was prevented. Ammonia and glucoseamine also inhibited HS3 synthesis and induced its decay. In these cases, only a combination of L-glutamate and P i could reverse the inhibition.

Ca2+ is a specific regulator of amino acid transport and protein synthesis in the water-mould Achyla

Abstract Transport of amino acids, uracil, thymidine, and Ca2+ in some cellulose-walled fungi is energy-mediated. Ca2+ enhances amino acid uptake and protein synthesis. Uracil and thymidine uptake and synthesis of RNA and DNA proceed independently of Ca2+ metabolism. The mucopolysaccharide dye ruthenium red (Ru(NH3)4(OH)Cl2) known to selectively inhibit the activity of a Ca2+ - stimulated Mg2+ - ATPase, specifically inhibits the transport of amino acids and Ca2+ in one wate-mould, Achyla . Saturation kinetics show that amino acids, ruthenium red, and Ca2+ may share close or common sites in the transport system for amino acids in these moulds.