Elastin peptides prepared from piscine and mammalian elastic tissues inhibit collagen‐induced platelet aggregation and stimulate migration and proliferation of human skin fibroblasts

We obtained pure elastin peptides from bovine ligamentum nuchae, porcine aorta, and bonito bulbus arteriosus. The inhibitory activity of these elastin peptides on platelet aggregation induced by collagen and the migratory and proliferative responsivenesses of human skin fibroblasts to these elastin peptides were examined. All of bonito, bovine, and porcine elastin peptides found to inhibit platelet aggregation, but bonito elastin peptides showed a higher inhibitory activity than bovine and porcine elastin peptides did. All elastin peptides enhanced the proliferation of fibroblasts 3.5‐ to 4.5‐fold at a concentration of 10 µg/ml. Bovine and porcine elastin peptides stimulated the migration of fibroblasts, with the optimal response occurring at 10^?1 µg/ml, while maximal response was at 10² µg/ml for bonito elastin peptides. Furthermore, pretreatment of fibroblasts by lactose depressed their ability to migrate in response to all elastin peptides, suggesting the involvement of elastin receptor in cell response. These results suggest that both mammalian and piscine elastin peptides can be applied as useful biomaterials in which elasticity, antithrombotic property, and the enhancement of cell migration and proliferation are required. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Demonstration of an endogenous, competitive inhibitor(s) of [3H] diazepam binding in bovine brain.

An endogenous inhibitor(s) of [³H] diazepam binding to synaptosomes has been demonstrated in bovine brain. The inhibitory activity of crude extracts is heat stable, dialyzable, and not affected by ether extraction. Three distinct peaks of inhibitory activity were resolved using Sephadex G-25 chromatography. The lowest molecular weight peak (<700 daltons) had the highest specific inhibitory activity and its inhibition of [³H] diazepam binding was competitive. A similar low molecular weight fraction was not observed in either muscle or liver suggesting that it may be unique to brain. Thin layer chromatography of the Sephadex G-25 fractions revealed a discrete band of inhibitory activity in the two low molecular weight peaks.     

Regulation of fibroblast proliferation in three-dimensional collagen gel matrix

Summary Fibroblastsin vivo reside in a three-dimensional (3-D) matrix. The 3-D culture method using collagen gels provides valuable information, but is also has some practical difficulties. In particular, the changes caused by the contraction of gels and the occasional abrupt detachment from the underlying surface have made extended culture difficult. In this study, the 3-D culture method was modified in order to observe the cells with minimal change of substrata for longer periods. The proliferation characteristics of fibroblasts cultured in gels in response to fetal calf serum (FCS), to two defined growth factors, insulin and platelet-derived growth factor (PDGF), and to a growth inhibitory factor, prostaglandin E₂ (PGE₂), were evaluated with this system in comparison with monolayer cultured fibroblasts. The DNA content of fibroblasts cultured both in gels and on dishes increased in response to FCS in a concentration-dependent manner. The proliferation of gel-cultured fibroblasts, however, was lower than that of dish-cultured cells, and higher concentrations of serum were necessary for proliferation. The response of gel-cultured cells to PDGF was also less than that of dish-cultured cells. In addition, fibroblasts cultured in gel culture did not respond to insulin, while the fibroblasts on dishes responded to insulin in a concentration-dependent manner. In contrast to the reduced response to growth stimulators, PGE₂ inhibited proliferation in gel culture and in monolayer culture similarly. The reduced responsiveness to growth stimulation but equivalent response to growth inhibition may account for reduced proliferation of fibroblasts in 3-D culture.     

Effects of fumonisin B1 alone and combined with deoxynivalenol or zearalenone on porcine granulosa cell proliferation and steroid production

Fumonisin B₁ (FB₁) is a Fusarium mycotoxin frequently occurring in corn in combination with deoxynivalenol (DON) and zearalenone. The aim of this study was to determine if FB₁, alone and combined with DON or α-zearalenol (ZEA), zearalenone major active metabolite, can affect granulosa cell proliferation, steroid production, and gene expression in swine. Porcine granulosa cells were cultured for 2 days in serum-containing medium followed by 1 or 2 days in serum-free medium with or without added treatments. Fumonisin B₁ had inhibitory effects on granulosa cell proliferation. Deoxynivalenol strongly inhibited cell growth, and no significant difference was detected in combination with FB₁. α-Zearalenol showed a stimulatory effect on granulosa cell numbers even in combination with FB₁. Regarding steroid production, FB₁ increased progesterone production, and FB₁ had no effect on estradiol production. Deoxynivalenol strongly inhibited progesterone and estradiol production, and FB₁ had no significant effect on this response. α-Zearalenol increased progesterone production, and its combination with FB₁ produced additive effects. α-Zearalenol had no effect on estradiol production, whereas it decreased estradiol production when co-treated with FB₁. Fumonisin B₁ was found to decrease CYP11A1 messenger RNA abundance, and the stimulatory effect of FB₁ on progesterone production was found to be not dependent on 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity suggesting that FB₁ increases progesterone production through a different mechanism. The results show that these Fusarium mycotoxins can influence porcine granulosa cell proliferation and steroid production, thereby demonstrating their potential reproductive effects on swine.     

Formation of 3H2O from [2-3H]- and [4-3H]estradiol by rat uteri in vitro: Possible role of peroxidase

The mitochondrial fraction of diethylstilbestrol-treated rat uteri, known to contain an estrogen-induced peroxidase, was able to catalyze the release of ³H₂O from either [2-³H]- or [4-³H]estradiol. Hydrogen peroxide added to this system increased the yield of ³H₂O but had no effect on mitochondrial preparations from ovariectomized rat uteri having only very low peroxidase activity. The reaction was inhibited by catalase and also occurred with lactoperoxidase in the presence of H₂O₂ but 2-hydroxyestradiol was not detected in any of these experiments. Under similar conditions, tyrosinase catalyzed the formation of the catechol estrogen with loss of ³H from [2-³H]- or [2,4,6,7-³H]- but not [4-³H]- or [6,7-³H]estradiol. It is proposed that the formation of ³H₂O from ³H-labeled estradiol in the estrogen-treated rat uterus may occur by a peroxidative mechanism which does not necessarily result in hydroxylation of the steroid.     

Effects of iron chelates on the transferrin-free culture of rat dermal fibroblasts through active oxygen generation

Summary Effects of nonchelating and chelating agents at 10 mM on the serum-free culture of rat dermal fibroblasts were investigated. A strong iron-chelating agent, iminodiacetic acid (IDA), and a weak one, dihydroxyethylglycine (DHEG), decreased iron permeation into preconfluent fibroblasts. A weak iron-chelating agent, glycylglycine (GG), a nonchelating agent, N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES), and human apotransferrin (10 μg/ml) increased the permeation with time. Iron may be essential for survival of fibroblasts because subconfluent fibroblasts exposed to 100 μM FeSO₄ in combination with transferrin, HEPES, or GG significantly decreased to release lactate dehydrogenase into the medium. Superoxide dismutase and dimethyl sulfoxide blocked the enzyme release, suggesting that superoxide and hydroxyl radical induce cellular damage but hydrogen peroxide (H₂O₂) generated by superoxide dismutation does not. GG significantly reduced H₂O₂ cytotoxicity. DHEG acted as a potent promoter of the iron-stimulated cellular damage if ascorbate or H₂O₂ was added to the medium. FeSO₄ and FeCl₃ (50 to 100 μM) individually combined with IDA maximally promoted fibroblast proliferation. Ascorbate increased formation of thiobarbituric acid-reactive substances from deoxyribose in the medium supplemented with FeSO₄ and either IDA or DHEG. Conversely, ascorbate decreased the formation in the medium with FeSO₄ and with or without other agents. Fibroblast proliferation may thus be stimulated through the active oxygen generation mediated by a redox-cycling between Fe³⁺ and Fe²⁺, which are dissolved in the medium at a high concentration, rather than through delivery of iron into the cells.     

Regulation of Pea Mitochondrial Pyruvate Dehydrogenase Complex : Does Photorespiratory Ammonium Influence Mitochondrial Carbon Metabolism?

Inactivation of the pyruvate dehydrogenase complex catalyzed by pyruvate dehydrogenase kinase was studied using intact mitochondria purified from green leaf tissue of pea (Pisum sativum L.) and dialyzed mitochondrial extracts. Thiamine pyrophosphate was inhibitory in dialyzed extracts but not in intact mitochondria, except in the presence of high concentrations of Na⁺. NH₄⁺, at concentrations as low as 20 micromolar, markedly stimulated inactivation in dialyzed extracts. K⁺ in the range 1 to 10 millimolar also enhanced inactivation. In contrast, Na⁺ was without affect at lower concentrations but was inhibitory at 10 to 100 millimolar levels. The effect of NH₄⁺ is discussed in relation to a possible regulatory interaction between photorespiratory NH₄⁺ production and the entry of carbon into the tricarboxylic acid cycle by way of the pyruvate dehydrogenase complex.     

Interleukin-1 and tumor necrosis factor are not synergistic for human synovial fibroblast PLA2 activation and PGE2 production

Patterns of arachidonic acid release and metabolism were altered in human synovial fibroblasts following exposure to cytokines. Recombinant interleukin-1 induced an approximate 3-fold in crease in [³H]-AA release, a 7-fold increase in PGE₂ production and a 2-fold increase in PLA₂ activity in human synovial fibroblasts. Recombinant tumor necrosis factor induced similar responses, however, the magnitude was less than that mediated by interleukin-1. A combination of the two cytokines had an additive effect on [³H]-AA release and PLA₂ activity while PGE₂ production was similar to that detected using interleukin-1 alone. [³H]-AA, was released in substantial amounts when sodium fluoride was used as a stimulus but PGE₂ was not. These data show that tumor necrosis factor and interleukin-1 can both activate synovial cell PLA₂ and induce generation of PGE₂, but act in an additive rather than a synergistic fashion. Furthermore, the data show that PGE₂ production is not always concordant with [³H]-AA release, suggesting that appropriate enzyme(s) must be activated.     

Comparison of the inhibitory effects of brain extract,high K+ and forskolin on juvenile hormone synthesis by Diploptera punctata corpora allata

We compared the effects of three treatments (brain extracts, high K⁺, forskolin) which inhibit JH synthesis by corpora allata of the cockroach Diploptera punctata incubated in vitro. Corpora allata showed a similar developmental sensitivity to all three treatments, with a high level of inhibition in glands of low activity. Inhibition of JH III synthesis by forskolin, high K⁺ or brain extract was antagonized by mevalonate and farnesoic acid, thus implicating an early biosynthetic step as the target of inhibition. All three treatments were also antagonized by Mn²⁺, suggesting that the inhibitory mode of action may be dependent upon Ca²⁺. Inhibition for forskolin and by high K⁺ was not only reversible, but caused a stimulation of JH synthesis upon removal of the inhibitor, suggesting that more than one regulatory mechanism is affected by these treatments.     

High resolution gel chromatography of proteins

An assay for the determination of l-glutaminase in extracts and intact cells is described. The method is based on the stereospecific release of ³H₂O from l-[2-³H]glutamine when l-glutaminase is coupled to l-aspartate:2-oxoglutarate amino transferase. The substrate, glutamine, and intermediate product, glutamate, are separated from the final reaction product, tritiated water, on a mixed-bed ion-exchange column which retains amino acids and organic acids but not water. The method has been adapted to determine the activity of l-glutaminase in cultured human diploid fibroblasts.     

A variety of Mytilus inhibitory peptides in the abrm of Mytilus edulis: Isolation and characterization

1. Five species of Mytilus inhibitory peptides, MIP_1–5, were isolated from acetone extracts of the anterior byssus retractor muscle (ABRM) of Mytilus edulis. MIP₁ and MIP₂ were shown to be S²-MIP and A²-MIP, respectively, first isolated from the pedal ganglia of the animal.2. All the five peptides had a common C-terminal structure of -Pro-Xaa-Phe-Val-NH₂, which was shown to be important for their biological activity.3. The five MIPs showed similar inhibitory effects on contractions of the ABRM but did not affect catch tension and its relaxation.4. In addition to the MIPs, catch-relaxing peptide (CARP) was also found in the ABRM.     

Anandamide targets aromatase: A breakthrough on human decidualization

In each menstrual cycle endometrial stromal cells (hESC) proliferate and differentiate into specialized decidual cells, a process termed decidualization, which regulates endometrial receptivity. Decidualization is mainly controlled by sex ovarian hormones, estradiol (E₂) and progesterone. E₂ plays an important role in the expression of the progesterone receptor and promotes the endometrial stromal cells differentiation. Our group previously reported that anandamide (AEA) impairs decidualization through cannabinoid receptor 1 (CB1). In this study, we hypothesized whether AEA inhibitory effect on cell decidualization could be mediated through interaction with aromatase and consequent interference in estradiol production/signaling. We used an immortalized human endometrial stromal cell line (St-T1b) and human decidual fibroblasts (HdF) derived from human term placenta. In cells exposed to a differentiation stimulus, AEA-treatment prevents the increase of the expression of CYP19A1 gene encoding aromatase, E₂ levels and of estradiol receptor expression, that are observed in differentiating cells. Regarding CYP19A1 mRNA levels, the effect was partially reverted by a CB1 receptor antagonist and by a COX2 inhibitor. In addition, we report that AEA presents anti-aromatase activity in placental microsomes, the nature of the inhibition being the uncommon mixed type as revealed by the kinetic studies. Structural analysis of the AEA-Aromatase complexes determined that AEA may bind to the active site pocket of the enzyme. In overall we report that AEA inhibits aromatase activity and may affect E₂ signaling crucial for the decidualization process, indicating that a deregulation of the endocannabinoid system may be implicated in endometrial dysfunction and in fertility/infertility disorders.     

Stimulation of the activity of prolyl hydroxylase in 3T3 fibroblasts by 1,10-phenanthroline

In confluent cultures of 3T3 fibroblasts, incubated for 24 h with 1,10-phenanthroline at 10^?5–10^?9 M, the activity of prolyl hydroxylase was significantly increased. 1,10-Phenanthroline was inhibitory at concentrations greater than 10^?4 M. The stimulatory effect of 1,10-phenanthroline manifets itself after 6 h inhubation and increased with time up to 48 h. 2,2′-dipyridyl and 5,6-dimethyl-1-1,10-phemamthroline were also stimulatory; a nonchelating analog, 1,7-phenanthroline had no effect.Cycloheximide did not modify the 1,10-phenanthroline effect. The stimulatory effect does not seem to depend on the shift of an inactive precursor of prolyl hydroxylase to an active form because 1,10-phenanthroline was shown to be ineffective in logarithmically growing cells.While dialysis of washed and homogenized cells significantly increased prolyl hydroxylase activity in cell extracts, undialyzed 1,10-phenanthroline treated samples exhibited higher prolyl hydroxylase activity than dialyxed controls.These data suggested to us that 1,10-phenanthroline and other chelating agents may be forming complexes with certain metal ions or protein-metal ions which are inhibitory towards prolyl hydroxylase.     

Rat Serum Factors Inhibiting the G1-S Transition In Hepatocytes

An attempt was made to detect the serum factors inhibiting the G₁-S transition in synchronized, baby rat hepatocytes. In untreated adult rat serum, this inhibitory activity was always linked to high molecular weight (HMW) compounds. Incubation of serum with trypsin or chymotrypsin resulted in the formation of a low molecular weight (LMW) G₁-S inhibitory factor. the same result was obtained with fractions from adult rat liver but not with kidney or spleen fractions. Separation of the LMW factor by ultrafiltration increased its specific activity by about 10³. the active period in the cell cycle of both the LMW and HMW factors was the same: the late G₁ phase. However, the activity of the LMW factor was not blocked by the Kunitz factor. an enzymatic transformation of the HMW factor might be induced by liver cell membrane-bound proteases and constitute a mechanism regulating hepatocyte proliferation.     

Effect of lysophospholipids on signaling in the Human jurkat T Cell Line

Lysophospholipids have recently been demonstrated to induce activation and proliferation of fibroblasts and other cell lineages by interacting with high affinity cell surface receptors leading to specific intracellular signaling events. Platelet activation, likely at the site of injury or inflammation, results in increased production of lysophospholipids suggesting a possible source of lysophospholipids. We have recently demonstrated that high concentrations of lysophospholipids are present in ascites and plasma from ovarian cancer patients, suggesting that physiologically produced lysophospholipids could interact with cells present in these fluids, including lymphocytes, and alter their function. We demonstrate herein that lysophosphatidic acid (LPA), lysophosphatidylserine (LPS), and sphingosylphosphorylcholine (SPC) activate the Jurkat T cell line. Each of the lysophospholipids induced a transient increase in cytosolic free calcium ([Ca2⁺]_i) in Jurkat cells. Increases in [Ca2⁺]_i were cross‐desensitized by LPA, LPS and SPC, suggesting that the lysophospholipids share the same receptor(s) or that their downstream signaling pathways converge or interact. Lysophosphatidylgycerol (LPG), a competitive inhibitor of the putative LPA receptor, inhibited the calcium releasing activity of LPA, but not that of LPS and SPC, suggesting that these lysophospholipids interact with different receptors and that desensitization is due to interactions in downstream signaling pathways. The ability of the lysophospholipids to induce increases in [Ca2⁺]_i was attenuated, but not completely blocked, by increases in [Ca2⁺]_i induced by activation of the thrombin receptor. In contrast, increases in [Ca2⁺]_i induced by the lysophospholipids and cross‐linking the CD3 component of the T cell receptor complex with the UCHT1 antibody did not undergo heterologous desensitization. Strikingly, LPA is sufficient to stimulate proliferation of Jurkat cells in serum‐free medium or in synergy with low concentrations of fetal bovine serum. In addition, LPA also increased the production of the T cell growth factor, interleukin 2 (IL‐2), by Jurkat cells treated with phorbol esters. LPS, in contrast, inhibited Jurkat proliferation while increasing IL‐2 production and SPC inhibited both processes. Thus, although all three lysophospholipids were sufficient to induce a transient increase in [Ca2⁺]_i in Jurkat cells, they induced markedly different physiological consequences. © 2005 Wiley‐Liss, Inc.     

Partial characterization of a mitogenic factor with somatomedin-like activity produced by culture WI-38 human fibroblasts

Conditioned serum-free medium (CSFM) obtained from WI-38 human fibroblasts was found to contain a mitogenic factor(s) with somatomedin (SM)-like activity. Treatment of the cells with cycloheximide eliminated the SM-like activity in CSFM, suggesting that these cells produce and release the activity. Gel filtration revealed that the fibroblast SM-like activity (FSLA) had a molecular size near 45,000. Isoelectric focusing of this FSLA yielded 2 bands of SM activity with pIs of 4.7 and 6.1, and corresponding molecular sizes of ～29,000 and 16,500, respectively. The FSLA obtained by gel filtration revealed parallel dose response curves with a basic SM in a SM radioreceptor and radioimmunoassay and stimulated: (1) ³⁵So₄ uptake by hypophysectomized rat cartilage; (2) (U-¹⁴C) glucose oxidation is isolated rat adipocytes; and (3) (³H) thymidine uptake and cell division in these same WI-38 fibroblasts. Out studies indicate that this FSLA and basic SM are similar but not identical.     

Effect of lysophospholipids on signaling in the human Jurkat T cell line

Lysophospholipids have recently been demonstrated to induce activation and proliferation of fibroblasts and other cell lineages by interacting with high affinity cell surface receptors leading to specific intracellular signaling events. Platelet activation, likely at the site of injury or inflammation, results in increased production of lysophospholipids suggesting a possible source of lysophospholipids. We have recently demonstrated that high concentrations of lysophospholipids are present in ascites and plasma from ovarian cancer patients, suggesting that physiologically produced lysophospholipids could interact with cells present in these fluids, including lymphocytes, and alter their function. We demonstrate herein that lysophosphatidic acid (LPA), lysophosphatidylserine (LPS), and sphingosylphosphorylcholine (SPC) activate the Jurkat T cell line. Each of the lysophospholipids induced a transient increase in cytosolic free calcium ([Ca²⁺]_i) in Jurkat cells. Increases in [Ca²⁺]_i were cross-desensitized by LPA, LPS and SPC, suggesting that the lysophospholipids share the same receptor(s) or that their downstream signaling pathways converge or interact. Lysophosphatidylgycerol (LPG), a competitive inhibitor of the putative LPA receptor, inhibited the calcium releasing activity of LPA, but not that of LPS and SPC, suggesting that these lysophospholipids interact with different receptors and that desensitization is due to interactions in downstream signaling pathways. The ability of the lysophospholipids to induce increases in [Ca²⁺]_i was attenuated, but not completely blocked, by increases in [Ca²⁺]_i induced by activation of the thrombin receptor. In contrast, increases in [Ca²⁺]_i induced by the lysophospholipids and cross-linking the CD3 component of the T cell receptor complex with the UCHT1 antibody did not undergo heterologous desensitization. Strikingly, LPA is sufficient to stimulate proliferation of Jurkat cells in serum-free medium or in synergy with low concentrations of fetal bovine serum. In addition, LPA also increased the production of the T cell growth factor, interleukin 2 (IL-2), by Jurkat cells treated with phorbol esters. LPS, in contrast, inhibited Jurkat proliferation while increasing IL-2 production and SPC inhibited both processes. Thus, although all three lysophospholipids were sufficient to induce a transient increase in [Ca²⁺]_i in Jurkat cells, they induced markedly different physiological consequences. © 1995 Wiley-Liss, Inc.     

In vitro cytostatic and immunomodulatory properties of the medicinal mushroom Lentinula edodes

Lentinula edodes, known as “shiitake” is one of the widely used medicinal mushrooms in the Orient. Antitumour activity of extracts of this mushroom has been widely demonstrated in animals and humans. However, this activity was shown to be host mediated and not by direct cytotoxic activity to cancer cells. This study demonstrates cytotoxic and cell growth inhibitory (cytostatic) effect of aqueous extracts of the mushroom on MCF-7 human breast adenocarcinoma cell line using an MTT cytotoxicity assay. Such effect was demonstrated with fruit body and mycelial extracts, the difference being that there was no significant suppression on normal cells with the latter. Furthermore mycelial extracts did not induce any cytostatic effect in both cancer and normal cell lines based on a DNA synthesis assay. The significant suppression of the proliferation of cancer cells was reflected by the comparatively low IC₅₀ values and the simultaneous higher respective values on normal fibroblast cells. The immunostimulatory activity of both fruit body and mycelial extracts was tested by the lymphocyte transformation test (LTT), which is based on the capacity of active immunomodulators to augment the proliferative response of rat thymocytes to T mitogens in vitro. Both fruit body and mycelial preparations were able to enhance the proliferation of rat thymocytes directly and act as co-stimulators in the presence of the T-mitogen PHA. Interestingly both extracts, similarly to zymosan showed SI_comit/SI_mit ratios of about 2, indicating adjuvant properties. Overall L. edodes aqueous extracts have demonstrated direct inhibition of the proliferation of human breast cancer cells in vitro and immunostimulatory properties in terms of mitogenic and co-mitogenic activity in vitro.     

In vitro anti‐enterovirus 71 activity of gallic acid from Woodfordia fruticosa flowers

Aims: The anti‐enterovirus 71 (EV71) activity of six Nepalese plants’ extracts and gallic acid (GA) isolated from Woodfordia fruticosa Kurz (family; Lythaceae) flowers were evaluated in Vero cells. Methods and Results: The anti‐EV71 activity of tested compounds was evaluated by a cytopathic effect reduction method. Our results demonstrated that flowers’ extracts of W. fruticosa exerted strong anti‐EV71 activity, with a 50% inhibitory concentration (IC₅₀) of 1·2 μg ml^?1 and no cytotoxicity at a concentration of 100 μg ml^?1, and the derived therapeutic index (TI) was more than 83·33. Rivabirin showed no antiviral activity against EV71. Furthermore, GA isolated from W. fruticosa flowers exhibited a higher anti‐EV71 activity than the extract of W. fruticosa flowers, with an IC₅₀ of 0·76 μg ml^?1 and no cytotoxicity at a concentration of 100 μg ml^?1, and the derived TI was 99·57. Conclusions: This study demonstrated that flower extracts of W. fruticosa possessed anti‐EV71 activity and GA isolated from these flowers showed stronger anti‐EV71 activity than that the extracts. Significance and Impact of the Study: Our results suggest that the GA from W. fruticosa flowers may be used as a potential antiviral agent.     

Hexokinase II of Pea Seeds

A second hexokinase (EC 2.7.1.1) was obtained from pea seed (Pisum sativum L. var. Progress No. 9) extracts. The enzyme, termed hexokinase II, had a high affinity (K_m, 48 micromolar) for glucose and a relatively low affinity (K_m, 10 millimolar) for fructose. The K_m for MgATP was 86 micromolar. Mg²⁺ was required for activity, but excess Mg²⁺ was inhibitory. MgADP inhibited hexokinase II. The addition of salts of monovalent cations increased hexokinase II activity. Al³⁺ was a strong inhibitor of the enzyme at pH 6.6 but not at the optimum pH (8.2). Citrate and 3-phosphoglycerate activated pea seed hexokinase II at pH 6.6, probably by coordinating with aluminum present as a contaminant in commercial ATP. The properties of hexokinase II are compared with those of the other three hexose kinases obtained from pea seed extracts. The possible role of these enzymes in plant carbohydrate metabolism is discussed.