Ingeborg A. Brand

Properties of a 19-kDa Zn2+-binding protein and sequence of the Zn2+-binding domains

Ribonuclease T1 was studied by two-dimensional 1H-NMR spectroscopy. Resonance assignments were obtained for the backbone protons of 95 amino acid residues and most of its side-chain protons using sequence-specific assignment procedures. The secondary structure elements of ribonuclease T1 were identified by an investigation of medium- and long-range nuclear Overhauser effects between the backbone and C beta protons. A low-resolution three-dimensional structure of ribonuclease T1 was deduced from qualitative interpretation of long-range nuclear Overhauser effects.

Inactivation of liver phosphofructokinase by phosphofructokinase phosphatase or by low pH is inhibited by fructose 2,6‐bisphosphate

Recent work from various laboratories [l-7] has shown that glucagon can affect the kinetic behaviour of rat liver phosphofructokinase (PFK). Although it had been concluded that this might result from a phosphorylation of the enzyme [4,5], Van Schaftingen et al. [6] have presented evidence that the effect of glucagon is mainly based on the decrease in the concentration of a low molecular weight l&and, later identified as fructose 2,6-bisphosphate [8]. The nature of the compound has been confirmed by Pilkis et al. [9]. Rat liver phosphofructokinase can be inactivated by an enzyme which exhibits the characteristic features of a phosphofructokinase phosphatase (removal of 32P-labelled inorganic phosphate from [32P]phosphofructokinase, requirement of Mg2+ [lo], inhibition by phosphate and inhibition by polyamines and polyarginine). Since we have found [ 1 l] that the PFK-phosphatase-catalyzed inactivation of purified rat liver phosphofructokinase could be inhibited by low concentrations of fructose 1,6_bisphosphate, we have examined in the present work the effect of fructose 2,6-bisphosphate on phosphofructokinase inactivation by PFK-phosphatase and on the spontaneous inactivation of phosphofructokinase at low pH. In addition we have studied the influence of fructose 2,6-bisphosphate on the inhibition by ATP and by citrate of purified rat liver phosphofructokinase, and compared the results with the effects of fructose 2,6-bisphosphate on purified rat liver L-type pyruvate kinase . The results show that fructose 2,6-bisphosphate is about SO-1000 times more active in comparison to fructose 1,6-bisphosphate on all parameters tested

The primary sequence of the PFK-1 inactivating zinc-binding protein as deduced from cDNA sequencing Identity of the zinc-binding protein with rat parathymosin

We have recently described the sequence of the Zn2+‐binding domain (43 amino acid residues) of a newly detected Zn2+‐binding protein which reversibly inactivates phosphofructokinase‐1 in a Zn2+‐dependent manner [(1986) J. Biol. Chem. 269, 5895–5900; (1988) Eur. J. Biochem. 177, 561–568]. Here, we describe the primary sequence of this protein based on a full‐length cDNA. A sequence comparison reveals the identity of the Zn2+‐binding protein with a protein called parathymosin‐α.

Metabolite‐controlled phosphorylation of hepatic phosphofructokinase proceeds by cAMP‐dependent protein kinase

In hepatocytes 32P‐incorporation into rat liver phosphofructokinase is stimulated by glucose as well as by glucagon, the effects of both stimuli being prevented by L‐alanine [Eur. J. Biochem. (1982) 122, 175]. The phosphopeptides of the enzyme derived from limited proteolysis by subtilisin and from exhaustive tryptic digestion were analyzed either by one‐dimensional mapping on sodium dodecyl sulphate‐polyacrylamide slab gels and by fingerprint mapping, respectively. It is shown that in vivo stimulation of 32P‐incorporation by glucose or by glucose plus glucagon results in identical phosphopeptide maps, and that these maps were identical with those obtained from phosphofructokinase phosphorylated in vitro with catalytic subunit of cAMP‐dependent protein kinase. It is concluded that in the intact liver cell phosphofructokinase is phosphorylated by cAMP‐dependent protein kinase but that the state of phosphorylation is modified by metabolite control.