Kohji Hasunuma

GTP-binding protein(s) in green plant, Lemnapaucicostata

GTP-binding protein(s) assayed by [35S]GTP gamma S was detected in the extract of Lemna paucicostata containing membrane components. About 15% of [35S]GTP gamma S activated GTP-binding protein, separated from unbound [35S]GTP gamma S by gel filtration, was retained on Millipore HAWP membrane filter. 80% of [35S]GTP gamma S activated protein, mixed with charcoal solution, remained in the supernatant after centrifugation of the mixture. Successive treatments of the assay mixture by charcoal solution established the assay system for the activation of GTP-binding protein. The Km value for the activation of GTP-binding protein was 8 nM.

GTP-Binding proteins in etiolated epicotyls of Pisum, sativum (Alaska) seedlings

Seven fractions of GTP-binding proteins separated by gel filtration of an extract of epicotyls of Pisum sativum seedlings were partially characterized. Seven fractions of GTP-binding proteins tentatively designated GP1 to GP7 had the capacity to be ADP-ribosylated by pertussis toxin. Pooled fractions of GP2 to GP7 showed Km values 2, 20, 50, 10, 3 and 1 nM, respectively. The binding of [35S]GTP gamma S to GTP-binding proteins was prevented competitively in the presence of 0.1 mM GTP and also prevented in the presence of 0.1 mM ATP. Binding of [35S]GTP gamma S to the proteins produced a decrease in their molecular weights.

Partial characterization and light-induced regulation of GTP-binding proteins in Lemna paucicostata.

Binding of GTP‐binding proteins with [35S]GTP7S in the extract containing membrane components of Lemna paucicostata 441 was inhibited by red or far red light by 20 to 25%, but blue light showed no or little effect. The plant used for the preparation of the extract was subjected to single darkness for 8 h, as both red and far red light inhibit flowering. The extract treated with 1% Lubrol was fractionated by gel filtration. Four species of GTP‐binding proteins, GL1, GL2, GL3 and GL4 were detected with Km values 3, 7, 80 and 4 nM, respectively. GL1, GL2 and GL3 were ADP‐ribosylated by pertussis toxin. The extract activated by [35S]GTP‐γS in darkness, under red light or under far red light was treated with 1% Lubrol and subsequent gel filtration of the extracts made it possible to detect GTP‐binding protein with a small molecular weight only in an extract labeled in darkness. The reduction in the molecular weight of GTP‐binding protein from the larger molecule associated with the binding of [35S]GTPγS was confirmed by rechromatography of the larger molecule activated by [35S]GTPγS in darkness. The binding of GL2 and/or GL3 with [35S]GTPγS was suggested to be inhibited by red or far red light.

Rapid change in cyclic 3′,5′-AMP concentration triggered by a light-off or light-on signal in Anabaena cylindrica

In a nitrogen-fixing cyanobacterium, Anabaena cylindrica, an immediate light-to-dark transition caused an increase in cAMP concentration within 1 min. Conversely, a dark-to-light transition caused a rapid fall in cellular cAMP concentration. The concentration of cyclic 3′5′-GMP(cGMP) was several times lower than that of cAMP and was not largely affected by a light-off or light-on signal.

Circadian oscillation and light-induced changes in the concentration of cyclic nucleotides in Neurospora

SummaryRhythmic oscillations in the concentration of cAMP in myeclia of wild type 74A, bd, bd frq-1 and bd frq-2 strains of Neurospora crassa grown in liquid media in darkness were detected. The period lengths of the rhythm of cAMP concentrations were about 21 to 22 h in 74A and bd, and 14≈20 and 19 h in bd frq-1 and bd frq-2. The concentration of cGMP oscillated slightly. In parallel experiments using solid medium, conidiation occurred about 22 h after the peak of cAMP concentration. Exposure of bd mycelia to white light (3.9 J/m2 ·s) at 7∼12 h after the onset of continuous darkness, when the concentration of cAMP was high, reduced the concentrations of CAMP and cGMP, whereas after 18 h of darkness when the concentration of CAMP was low, only the concentration of cGMP was reduced. The reduction in the concentrations of cAMP and cGMP by light occurred within 60 s. Exposure of mycelia to constant light resulted in an oscillation of cAMP concentration with a period length from 60 to 90 min. After 9 h of continuous darkness, exposure of bd mycelia to 0.5 mM cAMP or 0.5 mM cGMP for 1 h and subsequent transfer to solid media resulted in phase advances of the conidiation rhythm of 2.1 h and 1.2 h, respectively. Exposure to light for 1 h, however, caused a 4.3 h phase delay. These results strongly suggest that cAMP and possibly cGMP are factors controlling the circadian rhythm.

Partial characterization of GTP-binding proteins in Neurospora.

Six fractions of GTP-binding proteins separated by gel filtration of a mycelial extract containing membrane components of Neurospora crassa were partially characterized. [35S]GTP gamma S bound to GTP-binding protein was assayed by repeated treatments with a Norit solution and centrifugation. The binding of [35S]GTP gamma S to GTP-binding proteins was competitively prevented in the presence of 0.1 to 1 mM GTP but not in the presence of ATP. These GTP-binding proteins fractionated by the gel column had Km values of 20, 7, 4, 4, 80 and 2 nM. All six fractions of these GTP-binding proteins showed the capacity to be ADP-ribosylated by pertussis toxin.

Rhythmic oscillation of cyclic 3',5'-AMP and -GMP concentration and stimulation of flowering by cyclic 3',5'-GMP in Lemna paucicostata 381

Abstract— Rhythmic oscillation of the concentration of cyclic 3′,5′‐AMP and ‐GMP in a short day plant, Lemna paucicostata 381 in continuous darkness was detected after 2 cycles of 12 h dark and 12 h light entrainment. Cyclic 3′,5′‐AMP and ‐GMP, extracted from whole plant showed parallel oscillations in their concentrations for initial 36 h in continuous darkness and the oscillation in the concentration of cyclic 3′,5′‐AMP was roughly circadian. Their concentrations decreased during the initial 12 h (subjective night) and increased during 12 to 28 h. Exogenous addition of 2 μ.M of cyclic 3′,5′‐GMP or the dibutyryl derivative of it stimulated floral induction by 20 to 30%, when the plants were grown under 12 h light and 12 h dark regime. Cyclic 3′,5′‐AMP or the dibutyryl derivative of it showed little effect on flowering.

Characterization of cpd-1 and cpd-2 mutants which affect the activity of orthophosphate regulated cyclic phosphodiesterase in Neurospora

SummaryEnzymatic and genetic characterization ofcpd-1 andcpd-2, which exhibit rhythmic conidiation in liquid media and on solid media, were described with band (bd) strain as a reference.Cpd-1 andcpd-2 showed reduced growth in orthophosphate-free cyclic 3′,5′-AMP media, whilebd showed wild-type level of growth in the media. In low-phosphate media,cpd-1 andcpd-2 produced 19.2% and 9.8% of orthophosphate-regulated cyclic phosphodiesterase (cPDase) in culture media, while bd produced 123%. The intracellular levels of cPDase with Km of 1 × 10−5 M in high-phosphate media incpd-1, cpd-2 and bd were about 20%, 15%, and 10% of that in wild-type, respectively. In low-phosphate media, roughly equal levels of cPDase with Km of 1 × 10−5 M were produced in all strains, whereas the production of cPDase with Km of 2 × 10−3 M was reduced incpd-2, and that of cPDase with Km of 1 × 10−2 was reduced incpd-1 andcpd-2. The levels of intracellular cyclic 3′,5′-AMP incpd-1, cpd-2, andbd in high-phosphate media were 13.1%, 10.1%, and 69.6% of that in wild-type. Adenylate cyclase activity incpd-1, cpd-2, bd, andcr-1 was 69.3%, 34.0%, 63.2%, and 20.3% of that of wild-type (74A). The levels of Mg++-stimulated cyclic phosphodiesterase incpd-1, cpd-2, bd, andcr-1 at 0.2μM cyclic 3′,5′-AMP were 199%, 137%, 329%, and 293% of that of wild-type. It was suggested thatcpd-1, cpd-2, andbd are the genes controlling the levels of enzymes for cyclic 3′,5′-AMP.Cpd-I was mapped on LG IVR 19.4% distal topyr-2 andcpd-2 was mapped on LG IL 5.6% proximal toarg-1.

ATP‐GTP‐BINDING PROTEINS AND ENDOGENOUS ADP‐RIBOSYL TRANSFERASE IN Lemna paucicostata 441

Abstract— A crude extract containing membrane components of Lemna paucicostata was treated with 1% Lubrol PX and fractionated by gel nitration. Binding activities to non‐hydrolyzable analogues of ATP, [35S]ATPγS (adenosine 5′[;γ‐thio]triphosphate) and that of GTP, [35S]GTPγS (guanosine 5′[γ‐thiojtriphosphate) were detected in some fractions, and these activities were prevented in the presence of 0.1 mM ATP or GTP. ATP and GTP were 2 to 3 orders of magnitude more effective than CTP or UTP in preventing this binding activity. These fractions showed ATPase and GTPase activities with 1 nM [γ‐32P]ATP or [γ–32P]GTP substrate. Analyses by sodium dodecyl sulfate polyacrylamide gel electrophoresis of these fractions after binding with [35S]ATPγS or [35S]GTP‐γ S revealed that these fractions contained [35S]ATPγS and [35S]GTPγS binding proteins with molecular weights of 53 000 and 60 000, respectively. Both of these proteins were [32P]ADP‐ribosylated by endogenous ADP‐ribosyl transferase. Three proteins with molecular weights of 11 000, 12 000 and 13 000 which could bind [35S]ATP7S or [‐35S]GTP‐γ S were ADP‐ribosylated by endogenous ADP‐ribosyl transferase. Pertussis toxin stimulated ADP‐ribosylation of these proteins. Four proteins with molecular weight of 37 000, 50 000, 80 000 and 115 000 with PSS]ATP7S and [,3S]GTP7S binding activities were also detected. The signal transduction of light to underlying clock mechanism in Lemna may be controlled by ATP‐GTP‐binding proteins and by the ADP‐ribosylation of these proteins.

GTP-binding proteins in a cyanobacterium Anabaenacylindrica

GTP-binding proteins were detected in a crude extract containing membrane components of Anabaena cylindrica. The crude extract was treated with 1% Lubrol PX and was fractionated by gel filtration. The binding of [35S]GTP gamma S to GTP-binding proteins was prevented in the presence of 0.1 mM GTP and in the presence of 0.1 mM ATP. Six fractions of these GTP-binding proteins, tentatively designated GA1 to GA6, were ADP-ribosylated by pertussis toxin. GA3, GA4 and GA5 had Km values of 10, 60 and 7 nM, respectively. The molecular weights of some of these GTP-binding proteins were reduced after being labelled with [35S]GTP gamma S.