Membrane-bound geranylgeranyl diphosphate phosphatases: purification and characterization from Croton stellatopilosus leaves

Geranylgeranyl diphosphate phosphatase is an enzyme catalyzing the dephosphorylation of geranylgeranyl diphosphate (GGPP) to form geranylgeraniol (GGOH). The enzyme activity of GGPP phosphatase was detected in leaves of Croton stellatopilosus, a Thai medicinal plant containing plaunotol, a commercial anti-peptic acyclic diterpenoid. Enzymological studies of GGPP phosphatase in C. stellatopilosis leaves revealed that the enzyme is a membrane-bound protein that could be removed from 20,000g pellet by 0.1% Triton X-100 without significant loss of enzyme activity. The solubilized enzyme preparation was separated into two activity peaks, PI and PII, by BioGel A gel filtration chromatography. PI and PII were both partially purified and characterized. PI appeared to be a tetrameric enzyme with its native molecular mass of 232kDa and subunit size of 58kDa, whereas PII was a monomeric enzyme with a molecular mass of 30-34kDa. Both phosphatases utilized GGPP as the preferred substrate over farnesyl and geranyl diphosphates. The apparent K(m) values for GGPP of PI and PII appeared to be 0.2 and 0.1mM, respectively. Both activities were Mg(2+) independent and exhibited slightly acidic pH optima, 6.0-6.5 for PI and 6.5-7.0 for PII. The catalytic activities of PII was strongly inhibited by 1.0mM of Zn(2+), Mn(2+) and Co(2+), whereas that of PI was not affected. Both enzyme preparations were very stable upon storage at -20 degrees C for 45 days without significant loss of phosphatase activity. The presence of GGPP phosphatase enzymes in C. stellatopilosus is consistent with its putative involvement in the biosynthetic pathway of plaunotol although whether PI or PII is the actual enzyme involved in the pathway remains to be clarified.     

Inhibition and inactivation of glucose-phosphorylating enzymes from Saccharomyces cerevisiae by D-xylose

Three glucose-phosphorylating enzymes were separated from cell-free extracts of Saccharomyces cerevisiae by hydroxylapatite chromatography. Variations in the amounts of these enzymes in cells growing on glucose and on ethanol showed that hexokinase PI was a constitutive enzyme, whereas synthesis of hexokinase PII and glucokinase were regulated by the carbon source used. Glucokinase proved to be a glucomannokinase with Km values of 0.04 mM for both glucose and mannose. D-Xylose produced an irreversible inactivation of the three glucose-phosphorylating enzymes depending on the presence or absence of ATP. Hexokinase PI inactivation required ATP, while hexokinase PII was inactivated by D-xylose without ATP in the reaction mixture. Glucokinase was protected by ATP from this inactivation. D-Xylose acted as a competitive inhibitor of hexokinase PI and glucokinase and as a non-competitive inhibitor of hexokinase PII.     

Glucose repression in Saccharomyces cerevisiae is directly associated with hexose phosphorylation by hexokinases PI and PII

Genetic and biochemical analyses showed that hexokinase PII is mainly responsible for glucose repression in Saccharomyces cerevisiae, indicating a regulatory domain mediating glucose repression. Hexokinase PI/PII hybrids were constructed to identify the supposed regulatory domain and the repression behavior was observed in the respective transformants. The hybrid constructs allowed the identification of a domain (amino acid residues 102-246) associated with the fructose/glucose phosphorylation ratio. This ratio is characteristic of each isoenzyme, therefore this domain probably corresponds to the catalytic domain of hexokinases PI and PII. Glucose repression was associated with the C-terminal part of hexokinase PII, but only these constructs had high catalytic activity whereas opposite constructs were less active. Reduction of hexokinase PII activity by promoter deletion was inversely followed by a decrease in the glucose repression of invertase and maltase. These results did not support the hypothesis that a specific regulatory domain of hexokinase PII exists which is independent of the hexokinase PII catalytic domain. Gene disruptions of hexokinases further decreased repression when hexokinase PI was removed in addition to hexokinase PII. This proved that hexokinase PI also has some function in glucose repression. Stable hexokinase PI overproducers were nearly as effective for glucose repression as hexokinase PII. This showed that hexokinase PI is also capable of mediating glucose repression. All these results demonstrated that catalytically active hexokinases are indispensable for glucose repression. To rule out any further glycolytic reactions necessary for glucose repression, phosphoglucoisomerase activity was gradually reduced. Cells with residual phosphoglucoisomerase activities of less than 10% showed reduced growth on glucose. Even 1% residual activity was sufficient for normal glucose repression, which proved that additional glycolytic reactions are not necessary for glucose repression. To verify the role of hexokinases in glucose repression, the third glucose-phosphorylating enzyme, glucokinase, was stably overexpressed in a hexokinase PI/PII double-null mutant. No strong effect on glucose repression was observed, even in strains with 2.6 U/mg glucose-phosphorylating activity, which is threefold increased compared to wild-type cells. This result indicated that glucose repression is only associated with the activity of hexokinases PI and PII and not with that of glucokinase.     

Thermal inactivation of reduced ferredoxin (flavodoxin):NADP+ oxidoreductase from Escherichia coli

Ferredoxin (flavodoxin):NADP+ oxidoreductase (FNR) is an essential enzyme that supplies electrons from NADPH to support flavodoxin-dependent enzyme radical generation and enzyme activation. FNR is a monomeric enzyme that contains a non-covalently bound FAD cofactor. We report that reduced FNR from Escherichia coli is subject to inactivation due to unfolding of the protein and dissociation of the FADH(2) cofactor at 37 degrees C. The inactivation rate is temperature-dependent in a manner that parallels the thermal unfolding of the protein and is slowed by binding of ferredoxin or flavodoxin. Understanding factors that minimize inactivation is critical for utilizing FNR as an accessory protein for S-adenosyl-L-methionine-dependent radical enzymes and manipulating FNR as an electron source for biotechnology applications.     

Unfolding and inactivation of monomeric superoxide dismutase from E. coli by SDS

The inactivation and the unfolding of the naturally monomeric Cu, Zn, superoxide dismutase from E. coli upon addition of sodium dodecylsulphate have been studied. In contrast to the bovine enzyme, CD, EPR, NMR spectroscopy and pulsed low resolution NMR measurements found an unfolding transition followed by inactivation of the enzyme. During this transition the active site becomes accessible to the bulk water. The unfolding is reversible and both, the tridimensional structure of the protein and the active site, can be restored upon dialysis. In addition, unfolding occurs without loss of metals in the solution.     

Biosynthetic relationships between three rat apolipoprotein B peptides

Rat liver is unique in secreting very low density lipoproteins (VLDL) with three size-isoforms of apolipoprotein B: PI and PIII correspond to B-100 and B-48, respectively, while PII is slightly smaller than PI and has no counterpart in other species. Antibodies against a fusion protein corresponding to the extreme C-terminal region of PI fail to react with PII, suggesting that the latter lacks this moiety. [35S]Methionine-labeled perfused rat liver and isolated hepatocytes secrete labeled PII, but intracellular apoB contains only PI and PIII. The absence of labeled PII from Golgi VLDL, and the absence of continued PII production within the plasma compartment, strongly suggest that PIII-containing VLDL are formed by a one-time proteolytic processing of a certain proportion of PI-containing VLDL at the time of secretion. In contrast, polysome run-off translation experiments and analysis of polysome-bound nascent apoB chains show that both rat liver and intestinal polysomes release PIII-sized peptides directly at the appropriate point of elongation, in a manner incompatible with their formation by posttranslational processing. These results strongly suggest that the large (PI, B-100) and small (PIII, B-48) apoB peptides are translated from separate mRNAs. Thus, although both PII and PIII are C-terminally truncated products of PI, the mechanisms involved are entirely different.     

Proteolysis of hexokinase PII is not the triggering signal of carbon catabolite derepression in Saccharomyces cerevisiae

The role of hexokinase PII in mediating carbon catabolite derepression in yeast has been examined. Hexokinase isoenzyme PII (EC 2.7.1.1) was partially degraded when protease inhibitors were omitted from the buffer used for preparation of cell-free extracts. The hexokinase PII inactivation induced by D-xylose was correlated with derepression of maltase (EC 3.2.1.20) in the wild-type strain Saccharomyces cerevisiae G-517 and in D.308.3, a strain that contains the cloned hexokinase PII gene on a multicopy plasmid. This inactivation was not correlated with the loss of hexokinase PII protein as assayed by immunoblotting. We conclude that during the derepression process there is no release of proteolytic peptides from hexokinase PII.     

Cloning and restriction analysis of the hexokinase PII gene of the yeast Saccharomyces cerevisiae

Summary Carbon catabolite repression in yeast depends on catalytic active hexokinase isoenzyme PII (Entian 1980a). A yeast strain lacking hexokinase isoenzymes PI and PII was transformed, using a recombinant pool with inserts of yeast nuclear DNA up to 10 kbp in length. One hundred transformants for hexokinase were obtained. All selected plasmids coded for hexokinase isoenzyme PII, none for hexokinase isoenzyme PI, and carbon catabolite repression was restored in the transformants. Thirty-five independently isolated stable plasmids were investigated further. Analysis with the restriction enzyme EcoRI showed that these plasmids fell into two classes with different restriction behaviour. One representative of each class was amplified in Escherichia coli and transferred back into the yeast hexokinase-deficient strain with concomitant complementation of the nuclear mutation. The two types of insert were analysed in detail with 16 restriction enzymes, having 0–3 cleavage sites on transformant vector YRp7. The plasmids differed from each other by the orientation of the yeast insert in the vector. After yeast transformation with fragments of one plasmid the hexokinase PII gene was localised within a region of 1.65 kbp.     

Purification and kinetic properties of two soluble forms of calmodulin-dependent cyclic nucleotide phosphodiesterase from rat pancreas.

The calmodulin-dependent cyclic AMP phosphodiesterase and cyclic GMP phosphodiesterase (EC 3.1.4.17) activity of rat pancreas was purified 280-fold by affinity chromatography on calmodulin-Sepharose 4B. It then accounted for 15% of the total cytosol cyclic GMP nucleotide phosphodiesterase activity, in the presence of Ca2+, and represented a minor component of proteins specifically adsorbed by the column. This activity was resolved on a DEAE-Sephacel column into two fractions, termed PI and PII, on the basis of their order of emergence. After this step, PI and PII were purified 5650- and 3700-fold respectively. The molecular weight of PI was 175 000 and that of PII was 116 000, by polyacrylamide-gradient-gel electrophoresis. Both forms of phosphodiesterase could hydrolyse cyclic AMP and cyclic GMP, although PII displayed a higher affinity toward cyclic GMP than toward cyclic AMP. PI and PII exhibited negative homotropic kinetics in the absence of calmodulin. Upon addition of calmodulin, both enzymes displayed Michaelis-Menten kinetics and a 5-9-fold increase in maximal velocity, at physiological concentrations of cyclic GMP and cyclic AMP. When a pancreatic extract freshly purified by affinity chromatography was immediately analysed by high-performance gel-permeation chromatography on a TSK gel G3000 SW column, PII represented as much as 78% of the eluted activity. This percentage decreased to 52% when the sample was stored at 0 degrees C for 20 h before analysis, suggesting that PII, possibly predominant in vivo, was converted into the heavier PI form upon storage.     

Catalytic and regulatory properties of two forms of bovine heart cyclic nucleotide phosphodiesterase.

The soluble supernatant fraction of bovine heart homogenates may be fractionated on a DEAE cellulose column into two cyclic nucleotide phosphodiesterases (EC 3.1.4.-):PI and PII phosphodiesterases, in the order of emergence from the column. In the presence of free Ca2+, the PI enzyme may be activated several fold by the protein activator which was discovered by Cheung((1971) J. Biol. Chem. 246, 2859-2869). The PII enzyme is refractory to this activator, and is not inhibited by the Ca2+ chelating agent, ethylene glycol bis (beta-aminoethyl ether)-N, N'-tetraacetate (EGTA). The activated activity of PI phosphodiesterase may be further stimulated by imidazole or NH+4, and inhibited by high concentrations of Mg2+. These reagents have no significant effect on either the PII enzyme or the basal activity of PI phosphodiesterase. Although both forms of phosphodiesterase can hydrolyze either cyclic AMP or cyclic GMP, they exhibit different relative affinities towards these two cyclic nucleotides. The PI enzyme appears to have much higher affinities toward cyclic GMP than cyclic AMP. Km values for cyclic AMP and cyclic GMP are respectively 1.7 and 0.33 mM for the non-activated PI phosphodiesterase; and 0.2 and 0.007 mM for the activated enzyme. Each cyclic nucleotide acts as a competitive inhibitor for the other with Ki values similar to the respective Km values. In contrast with PI phosphodiesterase, PII phosphodiesterase exhibits similar affinity toward cyclic AMP and cyclic GMP. The apparent Km values of cyclic AMP and cyclic GMP for the PII enzyme are approx. 0.05 and 0.03 mM, respectively. The kinetic plot with respect to cyclic GMP shows positive cooperativity. Each cyclic nucleotide acts as a non-competitive inhibitor for the other nucleotide. These kinetic properties of PI and PII phosphodiesterase of bovine heart are very similar to those of rat liver cyclic GMP and high Km cyclic AMP phosphodiesterases, respectively (Russel, Terasaki and Appleman, (1973) J. Biol. Chem. 248, 1334).     

Cloning of hexokinase isoenzyme PI from Saccharomyces cerevisiae: PI transformants confirm the unique role of hexokinase isoenzyme PII for glucose repression in yeasts

Summary Hexokinase isoenzyme PI was cloned using a gene pool obtained from a yeast strain having only one functional hexokinase, isoenzyme PI. The gene was characterized using 20 restriction enzymes and located within a region of 2.0 kbp. The PI plasmid strongly hybridized with the PII plasmids isolated previously (Fröhlich et al. 1984). Hence there was a close relationship between the two genes, one of which must have been derived from the other by gene duplication. In conrrast, glucose repression was restored only in hexokinase PII transformants; PI transformants remained non-repressible. This observation provided additional evidence for the hypothesis of Entian (1980) that only hexokinase PII is necessary for glucose repression. Furthermore, glucose phosphorylating activity in PI transformants exceeded that of wild-type cells, giving clear evidence that the phosphorylating capacity is not important for glucose repression.     

Female sexual receptivity after partial copulations in the wolf spider (Schizocosa malitiosa)

Mated females of the wolf spider Schizocosa malitiosa are frequently refractory to further copulations. Copulation consists of two successive behavioural patterns: pattern I (PI) and pattern II (PII). During PI males make multiple consecutive insertions with each palp, and in PII males alternate the use of palps after each insertion until dismounting. As both patterns are inseminatory, another function – such as generating female reluctance – is suggested for this complex behaviour. Here we test experimentally whether female spiders mated only with PI, or only with PII, are reluctant to re-mate. Each copulating male was interrupted immediately after the end of PI (phase A), and the same male was instantly exposed to a second virgin female. After re-mounting, the male initially performed a brief recapitulation of PI followed by PII (both together considered as phase B). For testing female sexual reluctance, females were exposed to a second male 3 days after the partial mating, but whenever the mounting occurred it was interrupted. Females were raised and progeny was counted. Phase-A-mated females were more reluctant to re-mating than phase-B-mated females, but their respective progeny was similar. We suggest that female reluctance is caused by sperm-associated substances transferred during PI.     

Multistate folding of a hyperthermostable Fe-superoxide dismutase (TcSOD) in guanidinium hydrochloride: The importance of the quaternary structure

Superoxide dismutases (SODs), which are the first line of cellular defense against the toxic effects of reactive oxygen species, are metalloenzymes that catalyze the disproportionation of superoxide radicals to produce oxygen and hydrogen peroxide. Although much effort has been devoted to the folding mechanisms of Cu/Zn-SODs, little is known about the folding of Fe-SODs. In this research, the equilibrium unfolding and refolding of TcSOD, a tetrameric hyperthermostable Fe-SOD, were investigated by circular dichroism, intrinsic fluorescence, ANS fluorescence, size-exclusion chromatography and cross-linking experiments. The results herein suggested that the guanidine hydrochloride-induced unfolding of TcSOD involved a stable monomeric intermediate and a possible tetrameric intermediate. The Gibbs free energy of TcSOD dissociation was about 3-fold larger than that of the monomeric intermediate unfolding, which suggested that the quaternary structure plays a crucial role in TcSOD stability. A comparison of the thermodynamic parameters between TcSOD and other SODs also suggested that the stability of quaternary structure might be responsible for the hyperthemostability of TcSOD.     

Some Characteristics of Protein Kinases in Lemna paucicostata

The three protein kinases of Lemna paucicostata that are separableby DEAE-Sephacel chromatography have been designated PI, PIIand PIII [Kato et al. (1983) Plant & Cell Physiol. 24: 841].The optimum pH for the PI and PII enzymes was 7.5 and for thePHI enzyme 7.0. The activities of these enzymes were stimulatedby divalent cations, the maximum stimulation being producedby 5 nw Mg^{2 $} for PI, by 3 mM Co^{2 $} for PII and by 1 mM Mn^2$ for PIII. The cytokinins; benzyladenine, kinetin and zeatin,inhibited the activity of the PIII enzyme. The molecular weightsof the PI and PII enzymes did not change after incubation withcAMP even though their activities were regulated by this compound. (Received October 17, 1983; )     

Effects of arginine on rabbit muscle creatine kinase and salt-induced molten globule-like state

The arginine (Arg)-induced unfolding and the salt-induced folding of creatine kinase (CK) have been studied by measuring enzyme activity, fluorescence emission spectra, native polyacrylamide gel electrophoresis and size exclusion chromatography (SEC). The results showed that Arg caused inactivation and unfolding of CK, but there was no aggregation during CK denaturation. The kinetics of CK unfolding followed a one-phase process. At higher concentrations of Arg (>160 mM), the CK dimers were fully dissociated, the alkali characteristic of Arg mainly led to the dissociation of dimers, but not denaturation effect of Arg's guanidine groups on CK. The inactivation of CK occurred before noticeable conformational changes of the whole molecules. KCl induced monomeric and dimeric molten globule-like states of CK denatured by Arg. These results suggest that as a protein denaturant, the effect of Arg on CK differed from that of guanidine and alkali, its denaturation for protein contains the double effects, which acts not only as guanidine hydrochloride but also as alkali. The active sites of CK have more flexibility than the whole enzyme conformation. Monomeric and dimeric molten globule-like states of CK were formed by the salt inducing in 160 and 500 mM Arg H(2)O solutions, respectively. The molten globule-like states indicate that monomeric and dimeric intermediates exist during CK folding. Furthermore, these results also proved the orderly folding model of CK.     

The essential role of hypusine in eukaryotic translation initiation factor 4D (eIF-4D). Purification of eIF-4D and its precursors and comparison of their activities

Eukaryotic translation initiation factor 4D (eIF-4D) is the only protein known to contain the amino acid, hypusine [N epsilon-(4-amino-2-hydroxybutyl)lysine]. This unusual amino acid is formed post-translationally by modification of a single specific lysine residue in an eIF-4D precursor protein. Two separate eIF-4D precursors, each of which contains a lysine residue in place of the hypusine residue and each of which thereby serves as a protein substrate for the hypusine modification, were purified from DL-2-difluoromethylornithine-treated Chinese hamster ovary cells by means of a five-step procedure. These two precursors termed PI and PII both have apparent molecular masses of approximately 17 kDa, indistinguishable from that of eIF-4D, but exhibit more acidic isoelectric points (5.1 and 5.25 for PI and PII, respectively, compared with 5.37 for eIF-4D). These physical characteristics, together with other properties, indicate that eIF-4D differs from PII only in possessing the hypusine residue in place of a lysine residue, whereas an additional structural difference exists between PI and eIF-4D. eIF-4D from CHO cells provides a significant enhancement of methionyl-puromycin synthesis, a model assay for translation initiation. Neither PI nor PII stimulates this in vitro system. These findings are the first direct evidence that hypusine is essential for the biological activity of eIF-4D.     

Thermal denaturation of Bungarus fasciatus acetylcholinesterase: Is aggregation a driving force in protein unfolding?

A monomeric form of acetylcholinesterase from the venom of Bungarus fasciatus is converted to a partially unfolded molten globule species by thermal inactivation, and subsequently aggregates rapidly. To separate the kinetics of unfolding from those of aggregation, single molecules of the monomeric enzyme were encapsulated in reverse micelles of Brij 30 in 2,2,4-trimethylpentane, or in large unilamellar vesicles of egg lecithin/cholesterol at various protein/micelle (vesicle) ratios. The first-order rate constant for thermal inactivation at 45 degrees C, of single molecules entrapped within the reverse micelles (0.031 min(-1)), was higher than in aqueous solution (0.007 min(-1)) or in the presence of normal micelles (0.020 min(-1)). This clearly shows that aggregation does not provide the driving force for thermal inactivation of BfAChE. Within the large unilamellar vesicles, at average protein/vesicle ratios of 1:1 and 10:1, the first-order rate constants for thermal inactivation of the encapsulated monomeric acetylcholinesterase, at 53 degrees C, were 0.317 and 0.342 min(-1), respectively. A crosslinking technique, utilizing the photosensitive probe, hypericin, showed that thermal denaturation produces a distribution of species ranging from dimers through to large aggregates. Consequently, at a protein/vesicle ratio of 10:1, aggregation can occur upon thermal denaturation. Thus, these experiments also demonstrate that aggregation does not drive the thermal unfolding of Bungarus fasciatus acetylcholinesterase. Our experimental approach also permitted monitoring of recovery of enzymic activity after thermal denaturation in the absence of a competing aggregation process. Whereas no detectable recovery of enzymic activity could be observed in aqueous solution, up to 23% activity could be obtained for enzyme sequestered in the reverse micelles.     

Interaction of human recombinant alphaA- and alphaB-crystallins with early and late unfolding intermediates of citrate synthase on its thermal denaturation

We have investigated the role of recombinant human alphaA- and alphaB-crystallins in the heat-induced inactivation and aggregation of citrate synthase. Homo-multimers of both alphaA- and alphaB-crystallins confer protection against heat-induced inactivation in a concentration-dependent manner and also prevent aggregation. Interaction of crystallins with early unfolding intermediates of citrate synthase reduces their partitioning into aggregation-prone intermediates. This appears to result in enhanced population of early unfolding intermediates that can be reactivated by its substrate, oxaloacetate. Both these homo-multimers do not form a stable complex with the early unfolding intermediates. However, they can form a soluble, stable complex with aggregation-prone late unfolding intermediates. This soluble complex formation prevents aggregation. Thus, it appears that the chaperone activity of alpha-crystallin involves both transient and stable interactions depending on the nature of intermediates on the unfolding pathway; one leads to reactivation of the enzyme activity while the other prevents aggregation.     

Evidence for different requirements in physical state for the interaction of lipopolysaccharides with the classical and alternative pathways of complement

The influence of the state of aggregation of lipopolysaccharides upon their ability to interact with serum complement via either the classical or alternative pathway was studied. The anticomplement properties of two chromatographically distinct fractions of a phenol-extracted lipopolysaccharide isolated from Serratia marcescens were assessed by means of the standard sheep erythrocyte hemolytic assay and an alternative pathway-selective kinetic assay using rabbit erythrocytes. Both the high molecular weight PI fraction and the lower molecular weight PII fraction exerted anti-complement activity as determined in the sheep erythrocyte assay. Conversion of fractions PI and PII to their more soluble triethylamine salt forms resulted in a decrease in sedimentation coefficients and a corresponding loss of anticomplement activity. Further, the anticomplement activity of fractions PI and PII in the sheep erythrocyte assay was inhibited by polymyxin B, indicating a role for the lipid A region. Unlike the PII fraction, only the PI fraction can activate serum complement via the alternative pathway. This activity is not inhibited by polymyxin B, indicating that the response is not lipid A-mediated. Significantly, solubilization of the PI fraction with triethylamine had no effect on its ability to activate the alternative pathway. These studies clearly demonstrate that the interaction between lipopolysaccharides and serum complement is influenced by the state of lipopolysaccharide aggregation. However, this appears to be the case for lipopolysaccharide activation of the classical pathway but not of the alternative pathway.     

Ammo-Terminal Amino Acid Sequences of Pea Phytochrome II Fragments Obtained by Limited Proteolysis

Apical shoot of pea seedling contains two immunochemically distinctspecies of phytochrome, type I (PI) and type II (PII) [Abe etal. (1985) Plant Cell Physiol. 26: 1387-1399]. A PII samplewas prepared from crude extracts from apical shoot of 7-day-old,light-grown pea (Pisum sativum L. cv. Alaska) seedlings, usingbrushite, DEAE-agarose and an agarose linked monoclonal anti-peaPII antibody (mAPll) column chromatography. More than 75% ofthe PII sample consisted of one major 115 kDa chromopeptide,and less than 25% of PII was composed of two minor non-chromophoric49 and 48 kDa fragments, as judged by SDS-polyacrylamide gelelectrophoresis and zinc-induced chromophore fluorescence analysis.Limited proteolysis of PII was performed with either trypsinor Staphylococcus aureus V8 protease, and the resulting fragmentswere separated by SDS-polyacrylamide gel electrophoresis andblotted electrophoretically onto a polyvinylidene difluoridemembrane. Amino-terminal amino acid sequences of the four fragmentsof PII cut from the membrane were determined by a gas-phasesequencer. The sequence of the determined regions (76 aminoacid residues in total), reveals that PII was 63% homologouswith PI. Hence the primary structure of PII is distinct fromthat of PI. (Received July 21, 1989; Accepted September 1, 1989)