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The in vitro folding of rhodanese involves a competition between formation of properly folded enzyme and off-pathway inactive species. Co-solvents like glycerol or low temperature, e.g. refolding at 10 degrees C, successfully retard the off-pathway formation of large inactive aggregates, but the process does not yield 100% active enzyme. These data suggest that mis-folded species are formed from early folding intermediates. GroEL can capture early folding intermediates, and it loses the ability to capture and reactivate rhodanese if the enzyme is allowed first to spontaneously fold for longer times before it is presented to GroEL, a process that leads to the formation of unproductive intermediates. In addition, GroEL cannot reverse large aggregates once they are formed, but it could capture some folding intermediates and activate them, even though they are not capable of forming active enzyme if left to spontaneous refolding. The interaction between GroEL and rhodanese substantially but not completely inhibits intra-protein inactivation, which is responsible for incomplete activation during unassisted refolding. Thus, GroEL not only decreases aggregation, but it gives the highest reactivation of any method of assistance. The results are interpreted using a previously suggested model based on studies of the spontaneous folding of rhodanese (Gorovits, B. M., McGee, W. A., and Horowitz, P. M. (1998) Biochim. Biophys. Acta 1382, 120--128 and Panda, M., Gorovits, B. M., and Horowitz, P. M. (2000) J. Biol. Chem. 275, 63--70).

Folylpoly-gamma-glutamate synthetase (FPGS) catalyzes the activation of folate antimetabolites in mammalian tissues and tumors. We have determined the sequence, abundance, and function of human FPGS transcripts and found some striking differences to transcription of the mouse gene that allow production of FPGS isoforms in mouse liver and dividing tissues. Multiple human transcripts were identified, including the homolog of the mouse transcripts that initiate at two upstream exons. However, the human FPGS upstream promoter is infrequently used, and transcripts from this promoter include sequences homologous with only one of the upstream exons found in the mouse. The downstream promoter generates an array of transcripts, some of which do not produce active enzyme, a phenomenon not seen in the mouse. Hence, the dual promoter mechanism directing expression of FPGS isozymes in mouse tissues is not conserved in humans, and, unlike the mouse downstream promoter, the human downstream promoter is active in both dividing and differentiated tissues. This study raises questions about the differences in function served by the two mouse FPGS isozymes and how, or if, human tissues fulfill these functions. How humans and mice produce FPGS in only a subset of tissues using such different promoter structures also becomes a central issue.

The ICE/CED-3 family of proteases (caspases) play a central role in the execution phase of apoptosis. These proteases are synthesised as precursor molecules that require processing at specific aspartate residues to produce the two subunits that comprise the active enzyme. The activation of some of these proteases has been shown to occur during apoptosis. Here we show that Nedd2/ICH-1 (caspase-2) is activated during apoptosis induced by a variety of apoptotic stimuli. This activation occurs very early upon treatment of cells with apoptotic agents and appears to precede the activation of CPP32 (caspase-3). The activation of Nedd2 was not seen in cells that are resistant to apoptosis. These observations suggest that Nedd2 is an early effector in the pathway leading to cell death. Our observations also lend weight to the hypothesis that a group of caspases containing long prodomains are the first to be activated in response to apoptotic signals and that they lie upstream of a second class of caspases such as CPP32 containing short or no prodomains.

The restriction endonuclease BanII catalyzes the cleavage of double-stranded DNA and recognizes the degenerate sequence 5'-GPuGCPyC-3'. The poly-linker of M13mp18 contains one such sequence, 5'-GAGCTC-3'. The three other possible sites recognized by the enzyme were prepared by site-directed muta-genesis. The substitution of phosphate groups by phosphorothioate residues at some positions within the various recognition sites had relatively little effect on the rate of cleavage of the DNA. However, when the DNA contained a phosphorothioate group at the site of cleavage the rate of linearization of the DNA was decreased by a factor of 9. Interestingly, DNA which contained an additional phosphorothioate internucleotidic linkage immediately 3'-outside the recognition site could not be linearized by the enzyme. The results indicate that an important contact between enzyme and substrate is perturbed by the presence of the sulfur atom at this position.

The specific enzymatic activity of renal gamma-glutamyltranspeptidase is decreased from control levels (0.86 unit-1 mg-1) to minimal values within 2 h postinjection of 100-g rats with acivicin, an irreversible inhibitor of the enzyme. The recovery of transpeptidase specific activity was followed from 2 to 24 h postinjection and the data were used to calculate the absolute rate constants for degradation (kd = 0.47 +/- 0.03 day-1) and synthesis (ks = 0.41 +/- 0.04 unit-1 mg-1 day-1). This corresponds to a half-life for the renal transpeptidase of 1.46 +/- 0.09 days and 99% recovery of the specific activity by 10 days postinjection. Recovery was followed for 14 days and closely approximates this theoretical curve. The data from control experiments designed to test for secondary effects of the drug, acivicin, show that neither the relative rate of synthesis nor apparent rate of degradation for either total protein or gamma-glutamyltranspeptidase is significantly altered by acivicin treatment of rats. The results also show that the acivicin-inhibited transpeptidase is not degraded differently than enzymatically active enzyme. The individual heterodimer subunits also exhibit similar apparent half-lives in both control and treated animals. Thus, recovery of renal gamma-glutamyltranspeptidase specific activity after acivicin treatment can be used in vivo to determine absolute values of ks and kd for this enzyme. These values have not been reported for any other constituent of the renal brush-border membrane.

The separation of the 12 SH central subunit, the 5 SE peripheral metallo subunit, and the 1.3 SE biotinyl carboxyl carrier protein which are formed on the dissociation of transcarboxylase has been accomplished by molecular sieving on Bio-Gel. The 12 SH and 5 SE subunits have been obtained in nearly homogeneous form as judged by the sedimentation velocity profiles and by acrylamide gel electrophoresis. The 1.3 SE carboxyl carrier protein has given less consistent results; sometimes a single band of molecular weight of approximately 12,000 is obtained on gel electrophoresis in sodium dodecyl sulfate but sometimes there is an additional band of lower molecular weight of approximately 11,000. This lower molecular weight component may result from limited proteolytic degradation in spite of efforts to prevent it. Two or more bands are obtained in the absence of dodecyl sulfate. This heterogeneity may result from aggregation. subunits. The most effective reconstitution is accomplished by a two-step process. First, the 1.3 SE carboxyl carrier protein and 5 SE metallo subunit are combined to form the 6 SE complex; then this product is combined with the 12 SH subunit to yield active enzyme. With a limiting amount of the 6 SE complex and an excess of the 12 SH subunit the resulting enzymatic activity is proportional to the concentration of 6 SE complex. Likewise, with an excess of the 6 SF, complex and the 12 SH subunit limiting, the enzymatic activity is proportional to the concentration of the 12 SH subunit. The maximum specific activities of the reconstituted 6 SE complex and the 12 SH subunit were approximately 60% and 50%, respectively of the value estimated for their specific activities in the native 18 S form of the enzyme with three peripheral 6 SE subunits. Because assay of the 6 SE complex is done using an excess of the 12 SH subunit, this may yield enzyme with a single peripheral subunit. The 6 SE subunit may be less effective in this form. In the case of the 12 SH subunit, the activity is most likely low because the reconstituted 6 SE complex does not contain the full complement of 1.3 SE biotinyl carboxyl carrier proteins. The carboxyl carrier protein provides the groups which link together the 5 SE peripheral subunits with the central subunit. Evidence is presented that the 1.3 SE biotinyl carboxyl carrier must first combine with the 5 SE subunit to assume a form which effectively provides the combining groups for association with the 12 SH subunit.

As a first step towards understanding how the zymogen structure of prochymosin contributes to the process by which active enzyme is produced, we altered the nucleotide sequence which encodes the amino-terminal (or propeptide) region of the protein. Of the two sites for autoproteolysis of prochymosin, one where pseudochymosin is formed at a pH of 2 and the other where chymosin is formed at pH 4-5, we changed the former by removing one codon and changing two other codons. This genetically modified prochymosin was proteolytically processed and activated normally at pH 4.5. However, at pH 2.0 we observed only partial activation of the zymogen and found no evidence of proteolytic processing. The properties of this engineered prochymosin suggest that zymogen activation does not require proteolysis and that the two different zymogen processing sites can function independently from one another.

The nature of the activable form of renin in the kidney and other tissues has not been clear. Its identification and isolation from kidney have been hampered by rapid activation due to high levels of proteases. Using a pepstatin-Sepharose column, which distinguishes inactive renin from the active enzyme, evidence was obtained for the presence of a totally inactive zymogenic precursor of renin in the pituitary, pineal, and other regions of hog brain. The precursor has an approximate molecular weight of 50,000 and conversion to the active enzyme causes reduction in molecular weight to 43,000. Conversion of this active enzyme to an active but high molecular weight form (60,000) was also observed when the pituitary extract was treated with thiol-blocking reagents. This result was interpreted to indicate the presence of a binding protein. This study has demonstrated that inactive renin zymogen is different from so-called active big renin, which is a complex of active renin and the binding protein.

Tartrate dehydrogenase from Pseudomonas putida has been purified and crystallized. The enzyme catalyzes the reversible, DPN-linked oxidation of mesotartrate and of l(+)-tartrate to oxaloglycolate; l(-)- and d(+)-malate are inactive as either substrates or inhibitors. The enzyme requires manganous ions and one of several monovalent cations for activity. Both qualitative and quantitative differences are found in the requirement for monovalent cation, depending on whether meso- or l(+)-tartrate serves as substrate. The active enzyme has a molecular weight of 145,000 and appears to be composed of four subunits, each of which has a molecular weight of approximately 36,800.

The mode of polymerization of rabbit muscle phosphofructokinase has been investigated at 10° over a protein concentration range of 0.4 to 20 mg per ml in phosphate buffer at pH 6 and pH 8 and at a single concentration of 5 mg per ml in the region of pH 6 to 8. At pH 8 the data suggest that there are several forms of the active enzyme of molecular weight about 360,000 which differ markedly in their mode of polymerization. At least two of these forms have been separated on this basis. One form polymerizes rapidly and reversibly to higher molecular weight forms in what may be an indefinite manner. A second form may polymerize to a dimer of the active enzyme while a third form appears not to undergo any polymerization. At pH 6 and at the lowest protein concentrations studied here the enzyme exists as an inactive form of about 160,000 molecular weight. This species can also polymerize, although in a very different manner than the enzyme at pH 8. At very high enzyme concentrations, the pH 6 material, which presumably represents a protonated species of the enzyme, polymerizes to an active enzyme of which the molecular weight is 360,000 or greater. Between pH 6 and 8 the sedimentation behavior reflects the distribution of enzyme in the protonated (pH 6) and the deprotonated (pH 8) forms.

Dissociation of transcarboxylase at pH 9.0 and low ionic strength results in subunits with s20,w values of ∼6 S, ∼5 S, and 1.3 S. The 1.3 S subunit, designated as the biotin-carboxyl carrier protein, contains all the biotin initially present in the enzyme. Acid treatment (pH 5) of these unresolved subunits brings about reincorporation of the 1.3 S subunit into a reconstituted ∼6 S biotin subunit which has been isolated by sucrose density gradient centrifugation. Dissociation in the presence of 20% glycerol at pH 8 and low ionic strength yields a ∼12 S subunit and a ∼6 S subunit to which the 1.3 S biotin-carboxyl carrier protein remains attached. The ∼12 S subunit and the native ∼6 S biotin subunit have been separated by density gradient centrifugation. Active enzyme has been formed from a combination of the ∼12 S subunit with either the native ∼6 S biotin subunit or the reconstituted ∼6 S biotin subunit.