Phosphofructokinase from the flight muscle of bumblebee was purified to homogeneity and its molecular and catalytic properties are presented. The kinetic behavior studies at pH 8.0 are consistent with random or compulsory-order ternary complex. At pH 7.4 the enzyme displays regulatory behavior with respect to both substrates, cooperativity toward fructose 6-phosphate, and inhibition by high concentration of ATP. Determinations of glycolytic intermediates in the flight muscle of insects exposed to low and normal temperatures showed statistically significant increases in the concentrations of AMP, fructose 2,6-bisphosphate, and glucose 6-phosphate during flight at 25 degrees C or rest at 5 degrees C. Measuring the activity of phosphofructokinase and fructose 1,6-bisphosphatase at 25 and 7.5 degrees C, in the presence of physiological concentrations of substrates and key effectors found in the muscle of bumblebee kept under different environmental temperatures and activity levels, suggests that the temperature dependence of fructose 6-phosphate/fructose 1,6-bisphosphate cycling may be regulated by fluctuation of fructose 2,6-bisphosphate concentration and changes in the affinity of both enzymes for substrates and effectors. Moreover, in the presence of in vivo concentrations of substrates, phosphofructokinase is inactive in the absence of fructose 2,6-bisphosphate.

During childhood, play contributes to the physical, emotional, cognitive and social development of infants and children and may enhance future mental health. The aim of this study was to examine the relationship between play performance factors and psychosocial problems in school-aged children. A total of 142 typical Spanish children aged 5 to 9 years were included. Play performance was measured with the My Child's Play questionnaire, while the Strengths and Difficulties Questionnaire was used to evaluate internalizing and externalizing problems. The findings showed that personal, environmental and activity factors of play performance were associated with psychosocial problems and prosocial behavior in children. Moreover, children with high psychosocial difficulties reported significantly poorer play performance. As executive functioning during play was the factor that was most strongly associated with internalizing and externalizing psychosocial difficulties, it is possible that executive functions have a decisive role on both social cognition and self-regulation during play performance.

Immunization of dog and rat high pure rabbit pulmonary angiotensin-converting enzyme elicited, in some individuals, antibodies that inhibited their own converting enzyme. Active immunization with an immunologically related enzyme is thus a plausible approach for developing biologically based inhibitors of enzymes that are either in or accessible to the circulation. Rabbit testicular peptidyldipeptide hydrolase was purified to homogeneity and found to be a considerably smaller (Mr approximately 100,000) glycoprotein than pulmonary converting enzyme (Mr approximately 140,000). The two enzymes differed at their amino- and carboxy-termini. However, they exhibited identical catalytic properties, and antibodies prepared against either inhibited both similarly. In competition radioimmunoassays, antibodies against the pulmonary enzyme preferred it to the testicular species, whereas those against the latter did not distinguish between the two molecules. The testicular isozyme thus resembles an internal part of the pulmonary polypeptide, which includes its active site. In a reticulocyte lysate, mRNA from the lungs of immature and mature rabbits comparably primed the synthesis of a polypeptide (Mr approximately 129,000) that reacted with anticonverting enzyme antibodies. In contrast, an immunoreactive species was programed only by mRNA from the testis of mature animals, and this protein was much smaller (Mr approximately 85,000). Maturation dependence and a shorter polypeptide chain, the regulatory and structural properties that distinguish the testicular isozyme, are thus each pretranslationally determined.

The specific activity, molecular weight and immunological behavior of pure dipeptidyl carboxypeptidase from rabbit seminal fluid were found to resemble the corresponding properties of the pulmonary rather than the testicular isozyme.

Rabbit testicular dipeptidyl carboxypeptidase activity was purified by a procedure exploiting its affinity for N-alpha-[1-(S)-carboxy-3-phenylpropyl]-L-lysyl-L-proline. The molecular, catalytic, and immunological properties of the testicular enzyme are presented and compared with the corresponding properties of pulmonary angiotensin-converting enzyme. Although catalytically similar and immunologically related to pulmonary dipeptidyl carboxypeptidase, the testicular enzyme has a molecular weight (100,000) which is lower by a factor of about one-third and differs in its NH2 and COOH termini. Furthermore, we present evidence that the testicular enzyme is not a post-translation product of the pulmonary type enzyme. These data suggest that testicular and pulmonary dipeptidyl carboxypeptidase are two distinct proteins which are catalytically similar and immunologically closely related.

The NH2-and COOH-terminal sequences of the angiotensin-converting enzymes from rabbit lung and testis have been determined using less than 0.6mg of each protein. They are: (NH2)Thr-Leu-Asp-Pro-Gly-Leu-Leu-Pro-Gly-Asp- and -(Phe, Tyr)-Ser-Leu-Ala(COOH) for the pulmonary enzyme; and (NH2)Arg-Arg-Val-Ser-Asn-Asn-Gln-Ser-Ser- and -(Phe, Ala)-Glu-Leu-Ser(COOH) for the enzyme from testis.

Fructose 1,6-bisphosphatase was synthesized in vitro using Poly(A)+ RNA isolated from rat and rabbit liver and the specific translation products were identified by immunoprecipitation using antibody raised against the individual purified proteins, followed by electrophoresis on polyacrylamide gels in the presence of sodium dodecyl sulfate. The in vitro synthesis products were found to have subunit molecular weights which were identical to the corresponding purified proteins; 36,000 and 40,000 for the rabbit and rat enzymes, respectively. The rat liver enzyme synthesized in vitro could be converted to a form with a molecular weight nearly identical to that of the rabbit liver enzyme by treatment with trypsin. The results demonstrate that the difference in molecular weight between rat and rabbit liver fructose 1,6-bisphosphatase subunits is not due to limited proteolytic modification of the rabbit liver enzyme during purification. It is proposed that the rabbit liver enzyme in vivo lacks the COOH-terminal phosphorylation site which is present on the rat liver enzyme.

The molecular weight of newly synthesized dipeptidyl carboxypeptidase (angiotensin-converting enzyme; peptidyldipeptide hydrolase, EC 3.4.15.1) polypeptide primed in a reticulocyte lysate by poly(A)-containing RNA from mature rabbit testis was only about 65% that of the immunologically related species programmed by pulmonary RNA. Furthermore, in contrast to the pulmonary RNA-dependent product, the synthesis of this testicular protein was not directed by RNA from testes of immature animals. These findings indicate that a shorter polypeptide chain and pubertal expression--the structural and regulatory properties that distinguish the testicular dipeptidyl carboxypeptidase isozyme--are determined pretranslationally.

S-Carboxymethylated chicken muscle aldolase was treated with cyanogen bromide to cleave the 4 methionyl bonds per subunit. Five homogeneous fractions were obtained designated fragments I-V. Fragment I was derived from the N-terminus and fragment II from the C-terminus of the enzyme. Reduction of the enzyme with NaB3H4 in the presence of dihydroxyacetone phosphate decreases the enzymatic activity by 90%. Fragment III contained the Schiff base-forming lysine residue since more than 83% of the radioactivity introduced by NaB3H4 reduction of aldolase-dihydroxyacetone phosphate was found in this fraction. A tryptic peptide of 27 amino acid residues containing the substrate-binding site was isolated. The gross molecular structure of aldolase A from chicken muscle indicates a high degree of homology with mammalian muscle aldolases.

Chicken liver fructose 1,6-bisphosphatase binds to blue dextran-Sepharose affinity columns and is eluted by AMP, an allosteric inhibitor of the enzyme. On the other hand, bumblebee fructose 1,6-bisphosphatase, which is not inhibited by AMP, does not bind to blue dextran-Sepharose. Chicken liver 1,6-bisphosphatase binds 3.6 mol of AMP/mol of enzyme, while the bumblebee enzyme binds no AMP. However, bumblebee fructose 1,6-bisphosphatase can be activated by subtilisin, indicating that it possesses a protease-sensitive region similar to that present in mammalian fructose 1,6-bisphosphatase.

Native chicken liver fructose-1,6-bisphosphatase (Fru-P2ase) can bind to blue dextranSepharose affinity column and is not displaced by its sugar-phosphate substrate; however; it is readily eluted by the inhibitor 5′-AMP. Treatment of Fru-P2ase with pyridoxal 5′-phosphate (pyridoxal-P) in the presence of the substrate, fructose 1,6-bisphosphate, followed by reduction with NaBH4 leads to the formation of active pyridoxal-P derivatives of the enzyme showing diminished sensitivity to AMP inhibitor. The modified enzyme does not bind to the affinity column. On the other hand, in the presence of AMP modification of Fru-P2ase with pyridoxal-P occurs at the catalytic site; this modification does not alter its binding behavior toward the dye ligand. Blue dextran can also protect Fru-P2ase against AMP inhibition, and it is a competitive desensitizer for the nucleotide ligand. The results establish that blue dextran binds specifically to the allosteric site of the enzyme, and that the structure of this site may resemble that of the dinucleotide fold in other enzymes. Like native Fru-P2ase, digestion of pyridoxal-P-Fru-P2ase (with regulatory properties altered) with subtilisin causes a severalfold increase in the catalytic activity measured at pH 9.2, without significant change in the activity at pH 7.5, and produces a peptide with 56 amino acids. The residual subunit, Mr ~ 30,000, was found to contain all of the incorporated pyridoxal-P.

Fructose 1,6-bisphosphatase (Fru-P2ase,EC 3.1.3.11) purified from livers of fed rabbits has been reported to contain tryptophan, which is not present in the enzyme purified from livers of fasted animals. We now show that the tryptophan arises from small amounts of active or inactive rabbit liver aldolase in the Fru-P2ase preparations. Fru-P2ase free of tryptophan may conveniently be prepared by raising the temperature of the heat step in the purification procedure to 67 °C.

Multiple forms of phosphofructokinase in striated muscle and cardiac muscle of developing rabbit (Oryctolagus cuniculus) undergo changes with development, but not in brain and liver. The cardiac muscle of the 1-day-old rabbit contains phosphofructokinase A4 together with the four hybrid forms which were tentatively called A3C, A2C2, AC3, and C4. In older animals, phosphofructokinase C4 disappears first, followed by the hybrid forms, and only phosphofructokinase A4 persists in the adult animal. Both phosphofructokinase A4 and phosphofructokinase C4, as well as their hybrid forms, are present in developing embryonic brain and also in the brains of adult animals. Developing rabbit liver contains a single form of phosphofructokinase, but two isoenzymes are consistently seen in guinea pig liver. In striated muscle from fetal and 1-day-old rabbit, two isoenzymes are found, tentatively identified as A4 and the A3C hybrid. The results suggest that fetal phosphofructokinase A4 and phosphofructokinase C4, and their hybrids, might be present in striated muscle. Guinea pig tissues show a pattern of phosphofructokinase isoenzymes different from that in rabbit tissues.

A cyanogen bromide peptide, isolated from the NH2-terminus of rabbit liver FruP2ase, has been shown to have the following structure: Acetyl-Ala-Asp-Lys-Ala-Pro-Phe-Asp-Thr-Asp-Ile-Ser-Thr-Met-Thr-Arg-Phe-Val-Met. Previous evidence that the enzyme contains tryptophan located near the NH2-terminus and also that this tryptophan is lost on exposure of the enzyme to lysosomal fractions must be reevaluated. It is unlikely that the NH2-terminus would be reacetylated following proteolytic modification of this portion of the molecule.

Subtilisin catalyzes a limited modification of rabbit liver fructose 1,6-bisphosphatase (Fru-P2ase, EC 3.1.3.11), resulting in enhanced catalytic activity measured at pH 9.2. Sequence analysis of a cyanogen bromide peptide containing the subtilisin cleavage sites and of the NH2-terminal portion of the residual subunits shows that four peptide bonds in a limited region of the native molecule are susceptible to subtilisin. The cleavage sites are located at peptide bonds 57–58 (tyrosine-glycine), 60–61 (alanineglycine), 63–64 (threonine-asparagine), and 66–67 (threonine-glycine). Cleavage of the threonine-glycine bond appears to occur in about 60% of the peptide chains, and cleavage of the tyrosine-glycine bond occurs to a limited extent only at higher ratios of subtilisin to fructose bisphosphatase. The results suggest that the region of the molecule including residues 57–67 may exist as an exposed peptide susceptible to proteolytic attack. The residual subunits have been separated from the S-peptide (residues 1–60) and renatured in the presence of dithiothreitol. The reconstituted tetrameric enzyme lacking residues 1–60 is catalytically active. Thus, the NH2-terminal portion of the molecule is not required for this activity. The sequence of residues 1–78 of rabbit liver fructose 1,6-bisphosphatase has now been determined.

Digestion of rabbit liver fructose 1,6-bisphosphatase with subtilisin followed by denaturation of the protein yields a peptide containing 60 amino acid residues, including the blocked NH2-terminus. This peptide has the following sequence: Ac-Ala-Asp-Lys-Ala-Pr o-Phe-Asp-Thr-Asp-Ile-Ser-Thr-Met-Thr-Arg-Phe-Val-Met-Glu-Glu-Gly-Arg-Ly s-Ala-Gly-Gly-Thr-Gly-Glu-Met-Thr-Gln-Leu-Leu-Asn-Ser-Leu-Cys-Thr-Ala-Va l-Lys-Ala-Ile-Ser-Thr-Ala-Val-Arg-Lys-Ala-Gly-Ile-Ala-His-Leu-Tyr-Gly-Ile-Ala.

Digestion of rabbit liver fructose 1,6-bisphosphatase with subtilisin results in a several-fold increase in catalytic activity measured at pH 9.2. This change is due to cleavage of a peptide bond located 60 amino acid residues from the NH2-terminus. The S-peptide and the residual subunit appear as separate peptides in sodium dodecyl sulfate polyacrylamide gel electrophoresis and the S-peptide can be isolated by gel filtration in 9% HCOOH. Under nondissociating conditions, however, the S-peptide remains associated with the protein, and the tetrameric structure and original molecular weight are preserved. Thus the nicking of the peptide chain by subtilisin causes a conformation change that alters the catalytic properties of the enzyme.