Within the lysosomal cysteine protease family, cathepsin B is unique due to its ability to act both as an endopeptidase and a peptidyldipeptidase. This latter capacity to remove C-terminal dipeptides has been attributed to the presence of a 20-residue insertion, termed the occluding loop, that blocks the primed terminus of the active site cleft. Variants of human procathepsin B, where all or part of this element was deleted, were expressed in the yeast Pichia pastoris. A mutant, where the 12 central residues of the occluding loop were deleted, autoprocessed, albeit more slowly than the wild type proenzyme, to yield a mature form of the enzyme with endopeptidase activity comparable with the wild-type cathepsin B, but totally lacking exopeptidase activity. This deletion mutant showed a 40-fold higher affinity for the inhibitor cystatin C, suggesting that the occluding loop normally restricts access of this inhibitor to the active site. In addition, the binding affinity of the cathepsin B propeptide, which is a potent inhibitor of this enzyme, was 50-fold increased, consistent with the finding that the loop reorients on activation of the proenzyme. These results suggest that the endopeptidase activity of cathepsin B is an evolutionary remnant since, as a consequence of its membership in the papain family, the propeptide must be able to bind unobstructed through the full length of the active site cleft.

The effects of glucose, K+, and cholecystokinin octapeptide (CCK-8) on intracellular free Ca2+ concentration ([Ca2+]i) and membrane-associated protein kinase C (PKC) activity were examined in cultured islet cells from neonatal rats. Raising the glucose concentration from 2.8 to 22.2 mM or external K+ (from 5 to 45 mM), or adding CCK-8 (200 nM) all triggered a [Ca2+]i surge that peaked between 3 and 10 min afterward, depending on the stimulus, and then declined, either to a suprabasal plateau (glucose and K+) or to basal levels (CCK-8). These same manipulations triggered a burst of membrane-associated PKC activity that peaked between 5 and 10 min and then variously declined. Incubation in Ca2+-free medium abolished both the effects of glucose and K+ on [Ca2+]i and the stimulation of membrane-associated PKC activity. The K+-triggered stimulation of PKC activity was also inhibited by pretreating the cells with the general Ca2+ entry blocker lanthanum (1 mM). However, incubation in Ca2+-free medium did not affect the CCK-8-induced release Ca2+ from internal stores, although it abolished the burst of membrane-associated PKC activity, which showed the importance of Ca2+ influx as opposed to internal release for PKC activation. Thus, glucose, the principal stimulator of insulin secretion, rapidly stimulates Ca2+ influx into islet cells from neonatal rats, and it is probably this influx that stimulates membrane-associated PKC activity.

The effects of extracellular divalent cations on the intracellular Ca2+ concentration ([Ca2+]i) in neonatal rat islet cells were investigated to determine whether these cells, like several others, have signal-generating surface cation sensors. Raising the external Ca2+ concentration by 1 mM increments triggered either sustained increases in [Ca2+]i or large sharp [Ca2+]i spikes followed by return to a suprabasal level. The external Ca(2+)-triggered [Ca2+]i responses were abolished by treating the cells with the inhibitor of inositol phospholipid hydrolysis, neomycin (1.5 mM), but not by another phospholipase C inhibitor, U-73,122 (2.5 microM), or the voltage-sensitive Ca2+ channel blockers nifedipine (20 microM) and methoxyverapamil (D600; 50 microM). [Ca2+]i responses were also triggered by barium (Ba2+; 1 mM) and cobalt (Co2+; 1 mM). The Ba2+ responses were also inhibited by neomycin and unaffected by nifedipine or D600 and the Co2+ response required external Ca2+. Therefore, neonatal rat pancreatic islet cells may display divalent cation receptors/sensors on their surfaces. Activation of these putative receptors, which are coupled to neomycin-sensitive, voltage-independent, dihydropyridine-insensitive channels, by Ca2+, Ba2+ or Co2+ would trigger [Ca2+]i responses by opening these channels to admit external Ca2+ into the cell. The physiological function(s) of such cell-surface divalent cation receptors/sensors and the [Ca2+]i surges they generate in pancreatic islet cells is not known.