Inhibition of RNA Synthesis and the Relationship between Nucleolar and Mitotic Cycles in Zea mays Root Meristems

The relationship between nucleolar and mitotic cycles has beendetermined after treatment of root apices of Zea mays with ethidiumbromide. In the meristematic regions of the stele the two cyclesare not much displaced in relation to each other except fora delay in the onset of the disorganization phase. A few nucleolipersist into metaphase and a few nuclei undergo an amitoticdivision. In the cap initials the drug greatly delays the onsetof disorganization of the nucleolus, which normally occurs beforeprophase in this region. It also delays the completion of reorganizationso that fully organized nucleoli are no longer available duringthe last half of telophase. In the quiescent centre the onsetof disorganization and the end of reorganization of the nucleoliare also delayed in relation to mitosis. There is no evidencefor a delay in the onset of reorganization in any region ofthe meristem. Some cells form multiple micronucleoli and this aberrant behaviouroccurs more often in the cap initials than elsewhere as doesamitotic division.     

Dephosphorylation of phosphoproteins and synthetic phosphopeptides. Study of the specificity of the polycation-stimulated and MgATP-dependent phosphorylase phosphatases

The substrate specificity of different forms of polycation-stimulated (PCSH, PCSL, and PCSC) phosphorylase phosphatases and of the catalytic subunit of the MgATP-dependent protein phosphatase from rabbit skeletal muscle was investigated. This was done, with phosphorylase a as the reference substrate, using the synthetic phosphopeptides patterned after the phosphorylated sites of pyruvate kinase (type L) (Arg2-Ala-Ser(32P)-Val-Ala (S2), and its Thr(32P) substitute (T4)), inhibitor-1 (Arg4-Pro-Thr(32P)-Pro-Ala (T5), Arg2-Pro-Thr(32P)-Pro-Ala (T1), and its Ser(32P) substitute (S1)), and some modified phosphopeptides (Arg2-Ala-Thr(32P)-Pro-Ala (T2) and Arg2-Pro-Thr(32P)-Val-Ala (T3)), all phosphorylated by cyclic AMP-dependent protein kinase. In addition, casein(Thr-32P), phosphorylated by casein kinase-2, was also tested. The PCS phosphatases show a striking preference for the T4 configuration, PCSC being the least efficient. The catalytic subunit of the MgATP-dependent phosphatase was almost completely inactive toward all these substrates. As shown for the PCSH phosphatase, and comparing with T4, the two proline residues flanking the Thr(P) in T1 and T5, just as in inhibitor-1, drastically imparied the dephosphorylation by lowering the Vmax and not by affecting the apparent Km. The C-terminal proline (as in T2) by itself represents a highly unfavorable factor in the dephosphorylation. The critical effect of the sequence X-Thr(P)-Pro or Pro-Thr(P)-Pro (T1, T2, T5, and inhibitor-1) can be overcome by manganese ions. The additional finding that this is not the case with the Pro-Ser(P)-Pro sequence (S1) suggests that the effect of Mn2+ is highly substrate specific. These observations show the considerable importance of the primary structure of the substrate in determining the specificity of the protein phosphatases.     

Further characterization of mitochondrial protein phosphatase.

Mitochondrial protein phosphatase from rat liver exhibits rather wide substrate specificity, since it readily dephosphorylates, besides phosvitin, casein, and cytosol phosphoproteins, also ATP, ADP, inorganic pyrophosphate, p-nitrophenylphosphate.Aliphatic phosphate esters (β-glycerophosphate, glucose-6-P, serine-phosphate) are not dephosphorylated to any detectable extent.Evidence for the participation of a single enzyme in the dephosphorylation of phosvitin and ATP is provided. However, the different affinity toward the two substrates and other evidence suggest that the enzyme has in vivo the biological role of dephosphorylating, at least preferentially, the phosphoproteins.     

Endogenous proteinkinase-dependent phosphorylation of rat liver mitochondrial membranes

Isolated rat liver mitochondrial membranes are found to contain tightly bound protein substrate(s) which can be phosphorylated in the presence of ATP by protein kinase(s) previously extracted with 0.7 M NaCl from the membranes themselves and by phosvitin kinase purified from liver cytosol. Such proteinkinase-dependent phosphorylation, which seems to be cyclic AMP-independent, involves the seryl and threonyl residues of the protein substrate(s).